Tumor antigen

ABSTRACT

Tumor antigen inducing and/or activating HLA-A2-restricted tumor-specific cytotoxic T lymphocytes that is activated by recognizing HLA-A2 and a tumor antigen peptide, and a peptide or polypeptide derived from the tumor antigen, a polynucleotide encoding the peptide or a complementary strand polynucleotide thereof, a transformant comprising a recombinant vector which comprises the polynucleotide are provided.

FIELD OF THE INVENTION

[0001] The present invention relates to a tumor antigen, and moreparticularly relates to a peptide or a polypeptide recognized bytumor-specific cytotoxic T lymphocytes, a polynucleotide encoding thepeptide or the polypeptide and a complementary strand polynucleotidethereto, a recombinant vector comprising the polynucleotide, atransformant comprising the recombinant vector, an antibody against thepeptide or the polypeptide, a compound having any interaction with thepeptide or the polypeptide or the polynucleotide, a cytotoxic Tlymphocyte inducer consisting of the peptide and/or the polypeptide, anda pharmaceutical composition comprising the same, and a method forproducing the polypeptide, a method for screening for a compound havingany interaction with the peptide or the polypeptide or thepolynucleotide, a method for inducing cytotoxic T lymphocytes using thepeptide or the polypeptide, a method for measuring the peptide or thepolypeptide or the polynucleotide encoding the polypeptide, and areagent kit used for the measuring method.

BACKGROUND OF THE INVENTION

[0002] The immune system, particularly cytotoxic T lymphocytes (which,hereinafter, may be abbreviated to CTLs) play an important role in theexclusion of cancer in vivo. Infiltration of cytotoxic T lymphocytesexhibiting a cytotoxic activity against tumor cells has been detected atthe tumor site of a cancer patient (Arch. Surg., 126:200-205, 1990.) Atarget molecule (tumor antigen) of the tumor-specific cytotoxic Tlymphocytes was first discovered in a melanoma. A tumor antigengenerated in a tumor cell is degraded in the cell into a peptide (tumorantigen peptide) consisting of 8 to 11 amino acids, which binds to ahuman leukocyte antigen (HLA) molecule that is the majorhistocompatibility complex antigen to be displayed on the surface of thetumor cell. The cytotoxic T lymphocytes recognize a complex consistingof HLA and the tumor antigen peptide, and damage the tumor cell. Inother words, the cytotoxic T lymphocytes recognize the tumor antigenpeptide in an HLA-restricted manner.

[0003] HLA is a cell membrane antigen, and is expressed on almost alleukaryotic cells. HLA is mainly classified into class I antigen andclass II antigen. The HLA recognized by the cytotoxic T lymphocytestogether with an antigen peptide belongs to class I antigens. HLA classI antigens are further classified into HLA-A, HLA-B, HLA-C, and so on.It was reported that HLA has genetic polymorphism. The HLA-A2 allele,which is one of polymorphisms of HLA-A subregion, is found inapproximately 23% of African Blacks, approximately 53% of Chinese,approximately 40% of Japanese, approximately 49% of Northern Caucasians,and approximately 38% of Southern Caucasians.

[0004] As used herein, a tumor antigen means a protein, a polypeptide,or a peptide, which constitutes part of the tumor cell and is capable ofinducing tumor-specific cytotoxic T lymphocytes. A tumor antigen peptidemeans a peptide that is generated as a result of degradation of thetumor antigen in a tumor cell and can induce or activate tumor-specificcytotoxic T lymphocytes upon being expressed on the cell surface bybinding to an HLA molecule. In addition, the site of the amino acidsequence which is capable of inducing tumor-specific cytotoxic Tlymphocytes that is present in a tumor antigen is called a tumor antigenepitope (tumor antigen determinant.)

[0005] In recent years, many genes encoding tumor antigens that can berecognized by cytotoxic T lymphocytes have been identified from cDNA ofhuman tumor cells (Science 254:1643-1647, 1991; J. Exp. Med.183:1185-1192, 1996; J. Immunol. 163:4994-5004, 1999.) Some of thesegenes are involved in cellular proliferation and malignanttransformation, including HER/neu (Proc. Natl Acad. Sci. USA,92:432-436, 1995) mutant cdk (Science, 269:1281-1284, 1995) mutantCASP-8 (J. Exp. Med., 186:785-793, 1997) and so on.

[0006] On the other hand, a molecule such as a tumor rejection antigengene and a T cell antigen receptor (TCR), which are involved in specificimmunity, have been identified in melanoma, esophageal cancer, and othercancers in the past 10 years, and a specific immunotherapy of advancedcancer or metastatic cancer has been studied using the peptide.

[0007] Now, in Europe and in the United States, cancer vaccine therapyhas been developed in which cytotoxic T lymphocytes are activated by anadministration of a tumor antigen in a cancer patient. Results from aclinical test of a melanoma specific tumor antigen have been reported.For example, administration of a melanoma antigen gp-100 peptidesubcutaneously to melanoma patients along with administeringinterleukin-2(IL-2) intravenously gave a tumor regression in 42% of thepatients (Nature Medicine, 4:321, 1998.) In this way, by utilizing atumor antigen as a vaccine, an effective treatment against cancer can beachieved.

[0008] However, almost all of the identified tumor antigens are derivedfrom melanomas. Tumor antigens derived from epithelial cancers andadenocarcinomas, such as pancreatic cancer, which occur at highincidence rates, have been reported for such specific immunotherapy onlyin a few papers. Pancreatic cancer is one of the largest causes of deathby cancer in the world and causes about 27,000 deaths a year in the USAand about 50,000 deaths in Europe. The main factors causing these largenumbers of deaths are lack of an effective therapeutic method, thedifficulty of diagnosis, and the activity of this cancer. Only 1 to 4%of pancreatic cancer patients have overcome the disease, and theincidence substantially equals the death rate. Therefore, a new approachof therapy, for example, development of specific immunotherapy isneeded.

[0009] In addition, in view of the diversity of cancer, an identicaltumor antigen should not be expressed in the same degree in all cancercells. Naturally, cancer vaccine therapy by activating the cytotoxic Tlymphocytes using one kind of tumor antigen has a therapeutic effect oncancer having the tumor antigen. However, in order to induce andactivate the tumor antigen-specific cytotoxic T lymphocytes and obtain ahigh therapeutic effect corresponding to the diversity of cancer, it isimportant to discover and use many novel tumor antigens in accordancewith the diversity of cancer.

SUMMARY OF THE INVENTION

[0010] In one embodiment of the present invention, a peptide consistingof an amino acid sequence according to any one of those from SEQ ID NO:1 to SEQ ID NO: 44 is provided.

[0011] In another embodiment of the present invention, a polypeptideconsisting of an amino acid sequence according to any one of those fromSEQ ID NO: 45 to SEQ ID NO: 53 is provided.

[0012] In still another embodiment of the present invention, acomposition comprising one or more of peptides or polypeptides selectedfrom the peptides, which consist of the amino acid sequence according toany one of those from SEQ ID NO: 1 to SEQ ID NO: 44, and thepolypeptides, which consist of an amino acid sequence according to anyone of those from SEQ ID NO: 45 to SEQ ID NO: 53 is provided.

[0013] In an additional embodiment of the present invention, ananti-cancer vaccine comprising one or more of peptides or polypeptidesselected from the peptides, which consist of the amino acid sequenceaccording to any one of those from SEQ ID NO: 1 to SEQ ID NO: 44, andthe polypeptides, which consist of an amino acid sequence according toany one of those from SEQ ID NO: 45 to SEQ ID NO: 53 is provided.

[0014] In yet another embodiment of the present invention, ananti-cancer vaccine comprising one or more of peptides or polypeptidesselected from the peptides, which consist of the amino acid sequenceaccording to any one of those from SEQ ID NO: 1 to SEQ ID NO: 44, andthe polypeptides, which consists of an amino acid sequence according toany one of those from SEQ ID NO: 45 to SEQ ID NO: 53, for use in thetreatment of pancreatic cancer, colon cancer, or stomach cancer isprovided.

[0015] In another embodiment of the present invention, an inducer ofcytotoxic T lymphocytes comprising one or more of peptides orpolypeptides selected from the peptides, which consist of the amino acidsequence according to any one of those from SEQ ID NO: 1 to SEQ ID NO:44, and the polypeptides, which consist of an amino acid sequenceaccording to any one of those from SEQ ID NO: 45 to SEQ ID NO: 53 isprovided.

[0016] In still another embodiment of the present invention, a methodfor inducing cytotoxic T lymphocytes using one or more of peptides orpolypeptides selected from the peptides, which consist of the amino acidsequence according to any one of those from SEQ ID NO: 1 to SEQ ID NO:44, and the polypeptides, which consist of an amino acid sequenceaccording to any one of those from SEQ ID NO: 45 to SEQ ID NO: 53 isprovided.

[0017] In an additional embodiment of the present invention, apolynucleotide encoding a peptide or a polypeptide consisting of theamino acid sequence according to any one of those from SEQ ID NO: 1 toSEQ ID NO: 53, or the complementary strand thereof is provided.

[0018] In yet another embodiment of the present invention, apolynucleotide according to any one of those from SEQ ID NO: 54 to SEQID NO: 62, or the complementary strand thereof is provided.

[0019] In another embodiment of the present invention, a polynucleotideaccording to any one of those from SEQ ID NO: 54 to SEQ ID NO: 62,wherein a polypeptide encoded by the polynucleotide induces cytotoxic Tlymphocytes and/or is recognized by cytotoxic T lymphocytes, or thecomplementary strand thereof is provided.

[0020] In still another embodiment of the present invention, apolynucleotide that hybridizes to the polynucleotide or thecomplementary strand thereof under stringent conditions is provided.

[0021] In an additional embodiment of the present invention, arecombinant vector comprising the polynucleotide or the complementarystrand thereof or the polynucleotide that hybridizes to thepolynucleotide or the complementary strand thereof under stringentconditions is provided.

[0022] In yet another embodiment of the present invention, a recombinantexpression vector comprising the polynucleotide or the complementarystrand thereof or the polynucleotide that hybridizes to thepolynucleotide or the complementary strand thereof under stringentconditions is provided.

[0023] In another embodiment of the present invention, a transformanttransformed with the recombinant vector or the recombinant expressionvector, which comprises the polynucleotide or the complementary strandthereof or the polynucleotide that hybridizes to the polynucleotide orthe complementary strand thereof under stringent conditions is provided.

[0024] In still another embodiment of the present invention, a methodfor producing the polypeptide, which comprises culturing thetransformant transformed with the recombinant expression vector thatcomprises the polynucleotide or the complementary strand thereof or thepolynucleotide that hybridizes to the polynucleotide or thecomplementary strand thereof under stringent conditions is provided.

[0025] In an additional embodiment of the present invention, an antibodythat immunologically recognizes the peptide or the polypeptide isprovided.

[0026] In yet another embodiment of the present invention, a method forscreening for a compound that enhances at least recognition of thepeptide or the polypeptide by HLA-A2-restricted cytotoxic T lymphocytes,by interacting with the peptide or the polypeptide and/or HLA-A2 toenhance, and/or the compound that enhances expression of thepolynucleotide or the complementary strand thereof by interacting withthe same, is provided wherein the method uses at least one selected froma group consisting of the peptide, the polypeptide, the polynucleotide,the complementary strand thereof, the recombinant vector, therecombinant expression vector, the transformant, and the antibody.

[0027] On another embodiment of the present invention, a compoundobtained by the method for screening a compound that enhances at leastrecognition of the peptide or the polypeptide by HLA-A2-restrictedcytotoxic T lymphocytes, by interacting with the peptide or thepolypeptide and/or HLA-A2 to enhance, and/or the compound that enhancesexpression of the polynucleotide or the complementary strand thereof byinteracting with the same, is provided wherein the method uses at leastone selected from a group consisting of the peptide, the polypeptide,the polynucleotide, the complementary strand thereof, the recombinantvector, the recombinant expression vector, the transformant, and theantibody.

[0028] Instill another embodiment of the present invention, a compoundthat enhances recognition of at least one of the peptide or thepolypeptide by the HLA-A2-restricted cytotoxic T lymphocytes, or thecompound that enhances the expression of the polynucleotide or thecomplementary strand thereof by interacting with the same is provided.

[0029] In an additional embodiment of the present invention, apharmaceutical composition used for cancer treatment, comprising atleast one selected from a group consisting of the peptide, thepolypeptide, the polynucleotide, the complementary strand thereof, therecombinant vector, the recombinant expression vector, the transformant,the antibody, and the compound is provided.

[0030] In yet another embodiment of the present invention, use of thecomposition, the anti-cancer vaccine, the inducer of the cytotoxic Tlymphocytes, or the pharmaceutical composition for cancer disease isprovided.

[0031] In another embodiment of the present invention, a method formeasuring quantitatively or qualitatively the peptide or thepolypeptide, or the polynucleotide is provided.

[0032] In still another embodiment of the present invention, a reagentkit used in the method for measuring quantitatively or qualitatively thepeptide or the polypeptide, or the polynucleotide, is provided whereinthe kit comprises at least one selected from a group consisting of thepeptide, the polypeptide, the polynucleotide or the strain thereof, orthe antibody.

[0033] In an additional embodiment of the present invention, use of areagent kit for a test of the cancer disease is provided, wherein thekit is used to measuring quantitatively or qualitatively the peptide orthe polypeptide, or the polynucleotide, comprises at least one selectedfrom a group consisting of the peptide, the polypeptide, thepolynucleotide or the complementary strain thereof, or the antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034]FIG. 1 illustrates that OK-CTLp (HLA-A0207/A3101) lyses tumorcells in an HLA-A2-restricted manner.

[0035]FIG. 2 illustrates that recognition of the human pancreaticadenocarcinoma cell line Panc-1 by OK-CTLp and interferon-γ productionas a result thereof is an HLA-A2-restricted event.

[0036] FIGS. 3A-3B illustrate that OK-CTLp recognizes COS7 cells, inwhich each of cDNA clones 1 to 6 (FIG. 3A) and cDNA clone 7 (FIG. 3B),obtained from the human pancreatic adenocarcinoma cell line Panc-1, wascoexpressed with HLA-A2, in an HLA-A2-restricted manner.

[0037] FIGS. 4A-4F illustrate that cDNA clones 1 to 6, obtained from thehuman pancreatic adenocarcinoma cell line Panc-1, are recognized byOK-CTLp in a dose-dependent manner. FIGS. 4A-4F each show that OK-CTLprecognizes COS7 cells, in which each of cDNA clones 1 to 6, having highhomology with UBE2V, HNRPL, WHSC2, EIF4EBP1, ppMAPkkk, and 2-5 OAS3,respectively, was coexpressed with HLA-A2, in an HLA-A2-restrictedmanner. The symbol-▪-shows the amount of interferon-γ produced byOK-CTLp, when the HLA-A0207 gene was coexpressed with the each tumorantigen gene in target cells, -⋄-shows the amount of interferon-γproduced by OK-CTLp, when the HLA-A2402 gene was coexpressed with eachtumor antigen gene in the target cells.

[0038]FIG. 5 illustrates that OK-CTLp or OK-CTL clone recognizes fivepeptides derived from a gene product of the tumor antigen gene 1 that isobtained from the human pancreatic adenocarcinoma cell line Panc-1,which has high homology with UBE2V.

[0039]FIG. 6 illustrates that OK-CTLp or OK-CTL clone recognizes fourpeptides derived from a gene product of the tumor antigen gene 2 that isobtained from the human pancreatic adenocarcinoma cell line Panc-1,which has high homology with HNRPL.

[0040]FIG. 7 illustrates that OK-CTLp or OK-CTL clone recognizes fourpeptides derived from a gene product of the tumor antigen gene 3 that isobtained from the human pancreatic adenocarcinoma cell line Panc-1,which has high homology with WHSC2.

[0041]FIG. 8 illustrates that OK-CTLp or OK-CTL clone recognizes twopeptides derived from a gene product of the tumor antigen gene 4 that isobtained from the human pancreatic adenocarcinoma cell line Panc-1,which has high homology with EIF4EBP1.

[0042]FIG. 9 illustrates that OK-CTLp or OK-CTL clone recognizes threepeptides derived from a gene product of the tumor antigen gene 5 that isobtained from the human pancreatic adenocarcinoma cell line Panc-1,which has high homology with ppMAPkkk.

[0043]FIG. 10 illustrates that OK-CTLp or OK-CTL clone recognizes onepeptide derived from a gene product of the tumor antigen gene 6 that isobtained from the human pancreatic adenocarcinoma cell line Panc-1,which has high homology with 2-5 OAS3.

[0044] FIGS. 11A-11F illustrate representative peptides showing that thetumor antigen peptides, which are derived from products of the tumorantigen genes obtained from the human pancreatic adenocarcinoma cellline Panc-1, are recognized by OK-CTL clone in a dose-dependent manner.FIGS. 11A-11F show that OK-CTLp recognizes the peptides which arederived from gene products of the tumor antigen genes 1 to 6, havinghigh homology with UBE2V, HNRPL, WHSC2, EIF4EBP1, ppMAPkkk, and 2-5OAS3, respectively.

[0045] FIGS. 12A-12C illustrate that three peptides derived from a geneproduct of the tumor antigen gene 1 having high homology with UBE2V caninduce CTL showing cytotoxicity against HLA-A2⁺ tumor cell, fromperipheral blood mononuclear cells of a cancer patient. Thesymbol-▪-shows the human pancreatic adenocarcinoma cell line Panc-1(HLA-A0201/1101,)-♦-shows the human colon adenocarcinoma cell line SW620(HLA-A0201/2402,)-◯-shows the HLA-A2⁻ lung adenocarcinoma cell lineRERF-LC-MS (HLA-A1101/1101,)-Δ-shows an EBV transformed autologous Bcell (HLA-A0207/3101,) and-□-shows a PHA blast of autologous T cell(HLA-A0207/3101.) These symbols are also used in the same manner inFIGS. 13 to 17 described below.

[0046] FIGS. 13A-13B illustrate that two peptides derived from a geneproduct of the tumor antigen gene 2 having high homology with HNRPL caninduce CTL showing cytotoxicity against HLA-A2⁺ tumor cell, fromperipheral blood mononuclear cells of a cancer patient.

[0047] FIGS. 14A-14B illustrate that two peptides derived from a geneproduct of the tumor antigen gene 3 having high homology with WHSC2 caninduce CTL showing cytotoxicity against HLA-A2⁺ tumor cell, fromperipheral blood mononuclear cells of a cancer patient.

[0048] FIGS. 15A-15B illustrate that two peptides derived from a geneproduct of the tumor antigen gene 4 having high homology with EIF4EBP1can induce CTL showing cytotoxicity against HLA-A2⁺ tumor cell, fromperipheral blood mononuclear cells of a cancer patient.

[0049]FIG. 16 illustrates that one peptide derived from a gene productof the tumor antigen gene 5 having high homology with ppMAPkkk caninduce CTL showing cytotoxicity against HLA-A2⁺ tumor cell, fromperipheral blood mononuclear cells of a cancer patient.

[0050]FIG. 17 illustrates that one peptide derived from a gene productof the tumor antigen gene 6 having high homology with 2-5 OAS3 caninduce CTL showing cytotoxicity against HLA-A2⁺ tumor cell, fromperipheral blood mononuclear cells of a cancer patient.

[0051] FIGS. 18A-18E illustrate that tumor antigen peptides can induceCTL, which shows cytotoxic activity in an HLA-A2-restricted manner andin a dose-dependent manner, from peripheral blood mononuclear cells of acancer patient. FIGS. 18A-18E each show that peptides derived from geneproducts of the tumor antigen genes 1 to 5, having high homology withUBE2V, HNRPL, WHSC2, EIF4EBP1, and ppMAPkkk, respectively, induces CTLthat recognizes the peptide in a HLA-A2-restricted manner, fromperipheral blood mononuclear cells of a cancer patient. Thesymbol-▪-shows T2 cells in which the tumor antigen peptide was made toexpress and-⋄-shows autologous PHA blastoid T cells.

[0052] FIGS. 19A-19B illustrate that gene products of the tumor antigengenes KM-PA-2 (FIG. 19A) and KM-PA-4 (FIG. 19B), which were obtainedfrom the human pancreatic adenocarcinoma cell line CFPAC-1, arerecognized by OK-CTLp in an HLA-A2-restricted manner.

[0053] FIGS. 20A-20B show that peptides derived from the tumor antigengenes KM-PA-2 and KM-PA-4, respectively, which were obtained from thehuman pancreatic adenocarcinoma cell line CFPAC-1, can induce CTL fromperipheral blood mononuclear cells (PBMC) of a cancer patient, whichshows cytotoxicity against T2 cells which have been pulsed with thepeptide corresponding to the peptide used for stimulating the PBMC(left-hand of FIGS. 20A-20B,) and an HLA-A2⁺ tumor cell Panc-1(right-hand of FIGS. 20A-20B.)

[0054] FIGS. 21A-21C show that CTL, which was induced from peripheralblood mononuclear cells of a cancer patient by peptide derived from thetumor antigen gene KM-PA-2 obtained from the human pancreaticadenocarcinoma cell line CFPAC-1, lyses tumor cells in anHLA-A2-restricted manner.

DETAILED DESCRIPTION OF THE INVENTION

[0055] In order to identify a tumor rejection antigen gene and a tumorantigen encoded by the gene, which can be used for specificimmunotherapy for pancreatic cancer, HLA-A2-restricted tumor-specificcytotoxic T lymphocytes have been established in the present inventionthat are activated by recognizing HLA-A2 and a tumor antigen peptide(hereafter, this cell may be called OK-CTLp) from a colon cancerpatient, and genes encoding tumor antigens, which can be recognized bythese tumor-specific cytotoxic T lymphocytes (CTL,) have beenisolated/identified from a cDNA library of Panc-1 cell that as the humanpancreatic adenocarcinoma cell line using the gene expression cloningmethod. In the present invention, genes have also been identified, whichcan be recognized by CTL in the same way as described above, from genesidentified as those encoding the tumor antigen by the SEREX (Serologicalanalysis of recombinant cDNA expression libraries) method (Proc. NatlAcad. Sci. USA, 92:11910-11813, 1995.) In addition, based on the tumorantigen encoded by the gene obtained, a peptide having the tumor antigenepitope has been found in the present invention.

[0056] As used herein, a polypeptide means a long chain peptide ofarbitrary peptides comprising two or more amino acids bound to eachother by a peptide bond or by a modified peptide bond. For example, aprotein is included in the definition of polypeptide herein. Moreover, ashort chain peptide sometimes called an oligopeptide or an oligomer issimply called a peptide herein.

[0057] “Recognize” herein means that a subject distinguishes an objectfrom others and cognates it, for example, binds to the object cognized.Particularly, in the present invention, CTL which recognize the tumorcells or the tumor antigen peptides means that CTL binds through a Tcell receptor to the tumor antigen peptides that are presented by HLA.

[0058] “Activate” herein means to enhance or to make it work further athing or a state, which has an activity or an action. Particularly, inthe present invention, activation of CTL means that CTL recognizes anantigen being presented by HLA to produce IFN-γ or CTL showscytotoxicity against the target cells recognized by CTL.

[0059] “Induce” herein means to generate an activity or an action from athing or a state that are in a phase merely having the activity or theaction. Particularly, in the present invention, induction of anantigen-specific CTL means to make CTL, which specifically recognizes acertain antigen differentiate and/or proliferate in vitro or in vivo. Inaddition, the inducer of cytotoxic T lymphocytes in the presentinvention means a composition which changes the state, whereCD8-positive T lymphocytes specifically recognizing a certain antigen isabsent or present in a very low degree, to the state, where thecytotoxic T lymphocytes recognizing the antigen is present in a verydegree.

[0060] (Isolation and Identification of the Tumor Antigen Gene, TumorAntigen, and Tumor Antigen Peptide)

[0061] In the present invention, OK-CTLp that was HLA-A2-restrictedcytotoxic T lymphocyte described above was used as an effector cell andtumor antigens capable of activating this cell were isolated andidentified by using the gene expression cloning method. In other words,cDNA of the human pancreatic adenocarcinoma cell line Panc-1 and cDNA ofHLA-A0207 were cotransfected into COS-7 cells, and among those cells inwhich the transfected genes were expressed, the cells which enhanceIFN-γ production from OK-CTLp were selected, and hence, the geneencoding the tumor antigen capable of activating CTL was identified. Themethod will be presented in more detail in examples described herein. Asa result, seven cDNA clones were obtained which were recognized byOK-CTLp in an HLA-A2-restricted manner.

[0062] In addition, two genes, KM-PA-2 and KM-PA-4, encoding tumorantigens capable of activating CTL in an HLA-A2-restricted manner werefound in the same manner as described above from the genes encoding thetumor antigens, were detected from a cDNA library of the humanpancreatic adenocarcinoma cell line CFPAC-1 using the SEREX method andalready reported (Biochim. Biophys. Res. Commun., 281:936-944, 2001,).The SEREX method is a method that can be applied to detection of a tumorantigen. However, among 1500 or more kinds of tumor antigens detected bythis method, those being identified as tumor antigens capable ofinducing both cell-mediated immunity and humoral immunity are onlyMAGE-1, tyrosinase, and NY-ESO-1. Thus, the tumor antigen identified bythe SEREX method cannot always activate CTL. The present invention firstrevealed that the tumor antigen genes described above, KM-PA-2 andKM-PA-4, can activate CTL in an HLA-A2-restricted manner.

[0063] All seven cDNA clones described above, which were obtained fromPanc-1 cell, contained a complete open reading frame (ORF). Thenucleotide base sequence of these genes was determined by the Sangermethod (Chain Terminator method) to estimate the amino acid sequence onthe basis of the nucleotide base sequence. When a homology search wascarried out for these nucleotide base sequences and deduced amino acidsequences in an existing database such as GenBank, it was found thatthese genes were cDNAs whose nucleotide base sequences are novel, thoughthey have high homology with genes as described below. There is noreport on the known homologous genes functioning as a tumor antigen.With regard to clone 3 among the 7 cDNA clones (clones 1 to 7) obtained,the sequence of an initial clone, which was obtained by the geneexpression cloning method described above, and has high homology withthat of a gene of Wolf-Hirschhorn syndrome candidate 2 protein (WHSC2),was 25 bp shorter at the 5′-terminal region than that of WHSC2, so thatfull-length cDNA was obtained from the cDNA library of the Panc-1 cellby a standard colony hybridization method using the clone labeled with³²P as a probe. Hereinafter, the genes of the Panc-1 cell, from whichthe above-described clones 1 to 7 were derived, will be called gene 1 to7, respectively. Also, the polypeptides consisting of an amino acidsequence encoded by each gene are occasionally herein called geneproduct 1 to 7. Deduced amino acid sequences encoded by each of thesegenes are shown as SEQ ID NO: 45 to SEQ ID NO: 51, respectively, and thenucleotide base sequences thereof are shown as SEQ ID NO: 54 to SEQ IDNO: 60, respectively. The above described genes 1 to 6 and the gene 7were registered at the DNA Data Bank of Japan (DDBJ) of the NationalResearch Institute of Genetics in Jun. 12th, 2000 and Aug. 2, 2000,respectively (Table 1).

[0064] For KM-PA-2 and KM-PA-4, the homologous genes have been reportedas shown in Table 1 (Biochem. Biophys. Res. Commun., 281:936-944, 2001.The nucleotide base sequences of KM-PA-2 and KM-PA-4 are shown as SEQ IDNO: 52 and SEQ ID NO: 53, respectively, and the deduced amino acidsequences are shown as SEQ ID NO: 61 and SEQ ID NO: 62, respectively.TABLE 1 cDNA clone (gene) Polypeptide encoded by cDNA Base length (bp)Amino [DDBJ SEQ acid SEQ Homologous gene accession no.] ID: NO lengthID: NO [GenBank accession no.] 1 1775 54 PP 1 270 45ubiquitin-conjugated enzyme [AB044550] variant Kua (UBE2V) [AF155120] 22097 55 PP 2 589 46 heterogeneous nuclear [AB044547] ribonucleoprotein L(HNRPL) [NM_001533] 3 2243 56 PP 3 549 47 Wolf-Hirschhorn Syndrome[AB044549] candidate 2 protein (WHSC2) [AK001304] 4  831 57 PP 4 118 48eIF-4E-binding protein 1 [AB044548] (EIF4EBP1) [NM_004095] 5 2404 58 PP5 779 49 partial putatibe mitogen [AB044546] actibated protein kinasekinase kinase (ppMAPkkk) [AJ242724] 6 6707 59 PP 6 1087 502′,5′-oligoadenylate [AB044545] synthetase 3 (2-5 OAS3) [NM_006187] 7 769 60 PP 7 216 51 clevage and polyadenylation [AB046744] specificityfactor (CPSF) [U37012] KM-PA-2 2060 61 PP-KM-PA 2 634 52 KIAA0124 gene[D50914] [AB060694] human homologue of mouse block of proliferatin 1(Bopl) [BC005160] KM-PA-4 1841 62 PP-KM-PA 4 142 53 coactosin-likeprotein (CLP) [L54057]

[0065] The nucleotide base sequence of gene 1 has high homology withthat at of the ubiquitin-conjugated enzyme variant Kua gene (UBE 2V)registered at GenBank (accession no.AF155120.) The length of the deducedamino acid (aa) encoded by gene 1 was slightly longer than that encodedby the UBE2V gene (3 aa in positions 109 to 111.) The function of UBE2Vhas not so far been reported.

[0066] The nucleotide base sequence of gene 2 has high homology withthat of the heterogeneous nuclear ribonucleoprotein L (HNRPL) gene(accession no.NM_(—)001533.) However, the deduced amino acid length wasslightly longer than that of the HNRPL gene at the N-terminal positions1 to 31. The HNRPL gene product is a heterogeneous nuclearribonucleoprotein complex and provides a substrate for the processingevents that the mRNA precursor undergoes in the cytoplasm.

[0067] The nucleotide base sequence of gene 3 has high homology withthat of the Wolf-Hirschhorn syndrome candidate 2 proteins (WHSC2) generegistered at GenBank (accession no.AK001304.) The WHSC2 gene seems tohave some functions in the phenotype of WHS, a multiple malformationsyndrome characterized by mental and developmental defects resultingfrom a partial deletion of the short arm of chromosome 4.

[0068] The nucleotide base sequence of gene 4 has high homology withthat of the eIF-4E-binding protein 1 gene (EIF4EBP1, accessionno.NM_(—)004095.) The product of EIF4EBP1 gene is known as a translationinitiation factor that initiates insulin-dependent phosphorylation of4E-BP1, making it available to form an active cap-binding complex.

[0069] The nucleotide base sequence of gene 5 has homology with that ofthe partial putativemitogen-activatedprotein kinase kinase kinase gene(ppMAPkkk, accession no.AJ242724) registered at GenBank. The deducedamino acid sequence encoded by gene 5 is 230 aa longer at the N-terminaland 258 aa longer at the C-terminal as compared with that of theregistered ppMAPkkk gene (its function has not yet been reported.)

[0070] The nucleotide base sequence of gene 6 consists of 6707 bp andhas homology with that of the 2′,5′-oligoadenylate synthetase 3 gene(2-5 OAS3 gene, accession no.NM_(—)006187) with a total of 13-aadifferences at positions 18, 159, 249, 287, 288, 316, 393 to 398, and984. 2-5 OAS3 is known as an IFN-induced protein that plays an importantrole in immune-protection against microorganism infection.

[0071] The nucleotide base sequence of gene 7 has homology withpositions 3701 to 4463 of the Human cleavage and polyadenylationspecificity factor (CPSF, accession no.U37012) gene and the amino acidsequence is approximately 1226 aa shorter.

[0072] The nucleotide base sequence of gene KM-PA-2 has high homologywith that of the KIAA0124 and human homologues of mouse block ofproliferation 1 (Bop1.) The nucleotide base at position 1466 of theKIAA0124 gene is guanine (G) and thus, the amino acid residue of the465^(th) position is histidine (H). On the contrary, in gene KM-PA-2,they are adenine (A) and arginine (R), respectively.

[0073] The nucleotide base sequence of gene KM-PA-4 and the amino acidsequence encoded by the gene are identical to those of coactosin-likeprotein (CLP.)

[0074] In order to obtain tumor antigen peptides from theabove-described 9 genes encoding a tumor antigen, peptides were designedand synthesized based on the amino acid sequences encoded by theabove-described genes. For gene 7, the gene was regarded as a part of agene consisting of a longer base sequence, so that the peptides, whichwere derived from the amino acid sequence encoded by the gene (CPSF)homologous to gene 7, were also designed and synthesized. It has beenknown that a tumor antigen peptide capable of binding to the HLA-A2 hasa motif (a specific sequence) in its sequence. Then, at first, thepeptide having an HLA-A2 binding motif (a specific sequence) wassearched for in the literature (J. Immunol., 152:163, 1994;Immunogenetics, 41:178, 1994,) and the peptides of 9-mer to 11-mer,which were different from each other and suited to the motif obtained,were designed and synthesized based on the amino acid sequence encodedby the above described genes. Recognition of each peptide by CTL wasmeasured using OK-CTLp or several kinds of OK-CTL clones obtained bycloning OK-CTLp by the limiting dilution method using IFN-γ productionfrom these CTL as an indicator. The OK-CTL clones are the cellsrecognizing any one of the above-described genes 1 to 7. On the otherhand, OK-CTLp recognizes any of genes 1 to 7. The results revealed thatOK-CTLp is a cell population recognizing various kinds of tumorantigens. Therefore, when the OK-CTL clones were used to test thepeptide for its ability to activate CTL, a clone was used whichrecognizes the product of the gene encoding the same peptide as that tobe tested. Forty four peptides (SEQ ID NO: 1 to SEQ ID NO: 44; Table 2and Table 3) among those synthesized were recognized by OK-CTLp orOK-CTL clones and enhanced IFN-γ production from CTL. Among thesepeptides, P1 to P5, P6 to P9, P10 to P13, P14 and P15, P16 to P18, andP19 are the peptides consisting of a partial sequence of the amino acidsequences encoded by gene 1, gene 2, gene 3, gene 4, gene 5, and gene 6,respectively, and encoded also by genes UBE2V, HNRPL, WHSC2, EIF4-EBP1,ppMAPkkk, and 2-5 OAS3 having high homology with each genes. On theother hand, P25, P26, P27, P28, P30, and P31 are the peptides consistingof a partial sequence of the amino acid sequences encoded by gene 7 andgene CPSF having high homology with gene 7. P20, P21, P22, P23, P24,P29, and P32 consist of a partial sequence of the amino acid sequencespecific to CPSF. TABLE 2 SEQ ID Position of NO: in Origin of amino acidsequence Number peptide sequence Amino acid sequence listing P1 Gene 143-51 RLQEWCSVI 1 P2 (UBE2V) 64-73 LLLLARWEDT 2 P3 85-93 LIADFLSGL 3 P4201-209 LLQDWHVIL 4 P5 208-216 ILPRKHHRI 5 P6 Gene 2 140-148 ALVEFEDVL 6P7 (HNRPL) 404-412 CLYGNVEKV 7 P8 443-451 FMFGQKLNV 8 P9 501-510NVLHFFNAPL 9 P10 Gene3 103-111 ASLDSDPWV 10 P11 (WHSC2) 141-149ILGELREKV 11 P12 157-165 MLPLECQYL 12 P13 267-275 TLLRKERGV 13 P14 Gene4 51-59 RIIYDRKFL 14 P15 (EIF4EBP1) 52-60 IIYDRKFLM 15 P16 Gene 5290-298 QILKGLLFL 16 P17 (ppMAPkkk) 294-302 GLLFLHTRT 17 P18 432-440DLLSHAFFA 18 P19 Gene 6 666-674 QQLCVYWTV 19 (2-5 OAS3) P20 CPSF 285-293SLLYLNQSV 20 P21 CPSF 250-258 KVHPVIWSL 21 P22 CPSF 534-542 DMWTVIAPV 22P23 CPSF 882-890 QLGQGNLKV 23 P24 CPSF 392-400 LLLKYTEKL 24 P25 Gene 71367-1375 TMLPHHAGL 25 P26 Gene 7 1296-1304 LLRRADFHV 26 P27 Gene 71401-1409 ELLNRYLYL 27 P28 Gene 7 1358-1366 LLMLQNALT 28 P29 CPSF797-805 YQLPDWRLV 29 P30 Gene 7 1359-1368 LMLQNALTTM 30 P31 Gene 71358-1367 LLMLQNALTT 31 P32 CPSF 456-465 TQLATYSFEV 32

[0075] TABLE 3 SEQ ID NO: Position of in Origin of amino acid Amino acidsequence Number peptide sequence sequence listing P33 KM-PA-2 29-38LEWYDDFPHV 33 P34 115-124 FSGDVMIHPV 34 P35 172-180 WAQEDPNAV 35 P36179-188 AVLGRHKMHV 36 P37 326-335 RLWEVATARC 37 P38 348-356 VAWNPSPAV 38P39 585-593 DLLQNPLLV 39 P40 586-595 LLQNPLLVPV 40 P41 612-621VIFHPTQPWV 41 P42 KM-PA-4 15-24 NLVRDDGSAV 42 P43 57-65 RLFAFVRFT 43 P44104-113 VVQNFAKEFV 44

[0076] These tumor antigen peptides are presented on a cell surface byHLA-A2 and recognized by T cell receptor (TCR) expressed on thepeptide-specific OK-CTL clone. The peptides derived from gene 1 andUBE2V, gene 2 and HNRPL, or gene 6 and 2-5 OAS3 are recognized by OK-CTLclone expressing TCR-Vβ 8.1, TCR-Vβ 3.2, or TCR-Vβ 14. In addition,Peptides derived from gene 3 and WHSC2, gene 4 and EIF4EBP1, or gene 5and ppMAPkkk are recognized by OK-CTL clone expressing TCR-Vβ13,TCR-Vβ8.1, or TCR-Vβ18, respectively.

[0077] Two each of the OK-CTL clones that recognize peptides derivedfrom gene 1 and UBE2V, gene 2 and HNRPL, and gene 6 and 2-5 OAS3 wereexpressed TCR possessing different complementarity-determining regions 3(CDR 3; an element responsible for binding to antigenic epitopes on agroove of the HLA Class I molecules,) respectively. The OK-CTL clonerecognizing peptides derived from gene 3 and WHSC2, gene 4 and EIF4EBP1,and gene 5 and ppMAPkkk also expressed TCR possessing the differentCDR3, respectively.

[0078] Moreover, the above-described 44 peptides recognized by OK-CTLpeach induced HLA-A2-restricted tumor-specific CTL in vitro from theperipheral blood mononuclear cells (which, hereinafter, may beabbreviated to PBMC) which are autologous cells derived from a coloncancer patient from whom OK-CTLp was obtained, and/or from the PBMC ofHLA-A0201⁺ cancer patients (pancreatic cancer, colon cancer, and stomachcancer.) The above-described CTL induced from the peripheral bloodmononuclear cells of the cancer patients lysed HLA-A2⁺ tumor cells in adose-dependent manner. However,no lysis was observed in the HLA-A2⁻tumor cell RERF-LC-MS, and HLA-A⁺ autologous EBV-B cells and HLA-A⁺ Tcells stimulated by PHA, both of which are derived from normal cells. Inaddition, the above-described CTL showed cytotoxicity in adose-dependent manner against the T2 cells that were pulsed with thesame peptide as that used for stimulation.

[0079] On the basis of the examination described above, 44 tumor antigenpeptides capable of activating OK-CTLp were obtained. Furthermore, itwas found that these peptides could induce HLA-A2-restrictedtumor-specific CTLs from PBMC derived from patients of pancreaticcancer, colon cancer, and/or stomach cancer. In addition, it wasrevealed that both the pancreatic adenocarcinoma cell line Panc-1 andthe colon adenocarcinoma cell line SW620 are recognized by CTLp and byCTLs induced from PBMC by the above described peptide. This resultsuggests that pancreatic cancer and colon cancer have a common tumorantigen epitope recognized by host CTLs.

[0080] (Polypeptide and Peptide)

[0081] A polypeptide according to the present invention is a polypeptideconsisting of the amino acid sequence each of which is encoded by theabove described genes 1 to 7 obtained from the human pancreaticadenocarcinoma cell line Panc-1 or gene KM-PA-2 or KM-PA-4 obtained fromthe human pancreatic adenocarcinoma cell line CFPAC-1, and preferably, apolypeptide consisting of the amino acid sequence described in any oneof those from SEQ ID NO: 45 to SEQ ID NO: 53. These polypeptides can beused for inducing and/or activating CTL as a tumor antigen. Moreover,these polypeptides can be used as a material for specifying a tumorantigen epitope to obtain a tumor antigen peptide.

[0082] A peptide according to the present invention can be obtained bydesigning peptides, for example, which are suited for theHLA-A2-restricted motif, based on the amino acid sequence of theabove-described polypeptide, to select ones recognized by CTL, forexample, which activate and/or induce CTL, from the designed peptides. Apeptide according to the present invention may be the peptide having aproperty of a tumor antigen epitope presented on the surface of anantigen-presenting cell through binding to HLA-A2 and recognized by CTL.The peptide consists of amino acid residues of at least about 5 or more,preferably about 7 or more, and more preferably 9 to 10. Particularlypreferably, the peptide is one consisting of an amino acid sequencedescribed in any one of those from SEQ ID NO: 1 to SEQ ID NO: 44. Thesepeptides can be used as a tumor antigen peptide for activating and/orinducing the HLA-A2-restricted tumor-specific cytotoxic T lymphocytes.

[0083] For activating and/or inducing CTL, one of the above-describedpolypeptide or peptide may be used or they may be used in combination oftwo or more. As described above, CTL is a population consisting ofplural cells that recognize various antigens, so that it is recommendedto use these peptides preferably in combination of two or more.

[0084] A polypeptide or peptide, which has one or several amino acid(s)with a mutation such as deletion, substitution, addition, or insertionintroduced into the polypeptide or peptide specified as above and isrecognized by at least the HLA-A2-restricted CTL, is also includedwithin the scope of the present invention. A means for introducingmutations such as a deletion, substitution, addition, or insertion iswell known and, for example, Ulmer's technique (Science, 219:666, 1983)can be employed. When introducing such mutation, in view of preventing achange of the fundamental properties (such as the physical properties,activity, or immunological activity) of the peptide, mutual substitutionamong, for example, amino acids having similar properties (polar aminoacids, non-polar amino acids, hydrophobic amino acids, hydrophilic aminoacids, positively charged amino acids, negatively charged amino acids,aromatic amino acids, and so on) can be carried out. In addition, somemodification can be made on these peptides to such an extent that thereis no notable change of their function, such as modification of theconstitutive amino group or carboxyl group.

[0085] (Polynucleotide)

[0086] A polynucleotide according to the present invention is apolynucleotide consisting of the nucleotide base sequence described inany one of those from SEQ ID NO: 54 to SEQ ID NO: 62, which are thenucleotide base sequences of genes 1 to 7 or gene KM-PA-2 or KM-PA-4obtained from the human pancreatic adenocarcinoma cell line Panc-1 orthe human pancreatic adenocarcinoma cell line CFPAC-1 as describedabove, or the complementary strand thereof. The polynucleotide may alsobe a polynucleotide encoding each of the peptides consisting of theamino acid sequences described in any one of those from SEQ ID NO: 1 toSEQ ID NO: 44 or a polypeptide consisting of the amino acid sequencedescribed in any one of SEQ ID NO: 45 to SEQ ID NO: 53, or thecomplementary strand thereof. Moreover, the above-describedpolynucleotide may consist of a nucleotide base sequence of at leastabout 15 or more, preferably about 21 to 30 or more nucleotides, whereinthe nucleotide base sequence corresponds to a region encoding a tumorantigen epitope in the amino acid sequence of the polypeptides accordingto the present invention, or the complementary strand thereof. Selectionof a useful polynucleotide and determination of its nucleotide basesequence are possible, for example, by employing well-known proteinexpression systems to confirm the ability of the expressed protein toinduce and/or activate CTL.

[0087] Moreover, a polynucleotide that hybridizes to the above-describedpolynucleotide under stringent conditions is included in the scope ofthe present invention. In the case where the polynucleotide molecule isa DNA molecule, “a DNA molecule that hybridizes to a DNA molecule understringent conditions” can be obtained, for example, by the methoddescribed in “Molecular Cloning: A Laboratory Manual (Cold Spring HarborLaboratory, 1989.)” “To hybridize under stringent conditions” hereinmeans that a positive hybridizing signal is still observed even underthe condition in which, for example, hybridization is carried out in asolution containing 6×SSC, 0.5% SDS, and 50% formamide at 42° C. andthen, washing is carried out in a solution containing 0.1×SSC and 0.5%SDS at 68° C.

[0088] The above-described polynucleotide can induce and/or activate theHLA-A2-restricted CTL, when it is expressed in cells having HLA-A2. Inthis case, the above-described polynucleotide has a poly(A) structure inits 3′-terminal. The number of poly(A) does not have an influence on thesite encoding the amino acid acting as a tumor antigen, so that thenumber of poly(A,) of the polynucleotide is not limited.

[0089] All of the above-described polynucleotides provide geneticinformation useful for producing a polypeptide or a peptide according tothe present invention or can be also utilized as a reagent and astandard of a nucleic acid.

[0090] (Recombinant Vector)

[0091] A recombinant vector can be obtained by inserting theabove-described polynucleotide into an adequate DNA vector. The DNAvector used is properly selected in accordance with the kind of host andthe purpose of use. The DNA vector may be a naturally existing one andalso may be one that lacks a part of its DNA other than that necessaryfor replication. For example, vectors can be exemplified as thosederived from a chromosome, an episome, and a virus, for example, vectorsderived from a bacterial plasmid, derived from a bacteriophage, derivedfrom a transposon, derived from an enzyme episome, derived from aninserting element, and derived from an enzyme chromosome element, forexample, vectors derived from a virus such as baculovirus, papovavirus,SV40, vacciniavirus, adenovirus, fowlpox virus, pseudorabies virus, andretrovirus, and vectors prepared by combination of them, for example,vectors derived from the genetic element of the plasmid and thebacteriophage, for example, a cosmid and a phagemid. Further, anexpression vector and a cloning vector etc. can be used in accordancewith the desired purpose.

[0092] The recombinant vector, which comprises the constitutionalelements of the desired DNA sequence and a DNA sequence possessinginformation relating to replication and regulation, such as a promoter,a ribosome-binding site, a terminator, a signal sequence, an enhancer,and so on, can be prepared by combining them using well-known methods.As a method for inserting the polynucleotide according to the presentinvention into the above-described DNA vector, the well-known methodscan be employed. For example, a method can be used, wherein anappropriate restriction enzyme is chosen for treating a DNA to cleave itat a specific site, and then, the DNA is mixed with the DNA used as avector treated in the same way, followed by ligating with a ligase. Adesired recombinant vector can also be obtained by ligating an adequatelinker to the desired polynucleotide followed by inserting the resultantmolecule into a multi-cloning site of a vector suitable for a purpose.

[0093] (Transformant)

[0094] The DNA vector in which the above-described polynucleotide hasbeen inserted can be used to obtain a transformant by transforming awell-known host such as Escherichia coli, yeast, Bacillus subtillis, aninsect cell, or a mammalian cell therewith by well-known methods. In thecase of carrying out the transformation, a more preferable system isexemplified by the method for integrating the gene in the chromosome, inview of achieving stability of the gene. However, an autonomousreplication system using a plasmid can be conveniently used.Introduction of the DNA vector into the host cell can be carried out bystandard methods such that described in “Molecular Cloning: A LaboratoryManual” (ed. by Sambrook et al., Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1989.) Concretely, calciumphosphatetransfection, DEAE-dextran-mediated transfection, microinjection,cationlipid-mediated transfection, electroporation, transduction, scrapeloading, ballistic introduction and infection can be employed.

[0095] (Producing Polypeptide or Peptide)

[0096] Using an expression vector as a DNA vector for transduction ofthe above-described transformant, a polypeptide or a peptide accordingto the present invention can be provided. A transformant, transformedwith a DNA expression vector comprising the above-describedpolynucleotide, is cultured under well-known culture conditions suitablefor each host. Culturing may be conducted by using indicators, such as afunction of the polypeptide or a peptide according to the presentinvention that is expressed by the transformant, particularly at leastthe activity to induce and/or activate CTL, or the peptide or the amountof the peptide produced in the host or outside of the host. Subculturingor batch culturing may be also carried out using an amount of thetransformant in the culture as an indicator.

[0097] A peptide according to the present invention can be produced by ageneral method known in peptide chemistry. For example, “PeptideSynthesis (Maruzen) 1975” and “Peptide Synthesis, Interscience, NewYork, 1996” are exemplified. However, any widely known method can beused.

[0098] A polypeptide or peptide according to the present invention canbe purified and collected by a method, such as a gel filtrationchromatography, an ion column chromatography, an affinitychromatography, and the like, in combination, or by fractionation meanson the basis of a difference in solubility using ammonium sulfate,alcohol, and the like, using a CTL-activating ability of the polypeptideor the peptide as an indicator. More preferably used is a method,wherein the polypeptide or the peptides are specifically adsorbed andcollected by using polyclonal antibodies or monoclonal antibodies, whichare prepared against the polypeptide or the peptides based on theinformation of their amino acid sequences.

[0099] (Antibody)

[0100] An antibody according to the present invention is prepared byusing the above-described polypeptide or peptide as an antigen. Anantigen may be the above-described polypeptide or peptide itself, or itsfragment that is composed of at least 5, more preferably at least 8 to10 amino acids. In order to prepare the antibody specific to theabove-described polypeptide or peptide, a region consisting of the aminoacid sequence intrinsic to the above-described polypeptide or peptide isdesirably used. The amino acid sequence is not necessarily homologous tothe amino acid sequence of the polypeptide or the peptide, but ispreferably a site exposed to outside of a stereo-structure of thepolypeptide or the peptide. In such a case, it is sufficient that theamino acid sequence of the exposed site is consecutive in the exposedsite, even if it may be discrete in its primary structure. The antibodyis not limited as long as it binds or recognizes the polypeptide or thepeptide immunologically. The presence or absence of the binding or therecognition can be determined by a well-known antigen-antibody bindingreaction.

[0101] In order to produce an antibody, a well-known method for antibodyproduction can be employed. For example, the antibody is obtained byadministration of the polypeptide or peptide according to the presentinvention to an animal in the presence or absence of an adjuvant with orwithout linking such to a carrier so as to induce humoral immunityand/or cell-mediated immunity. Any carrier can be used as long as it isnot harmful to the host. For example, cellulose, a polymerized aminoacid, albumin, and the like are exemplified, but not limited thereto. Asan animal used for immunization, a mouse, rat, rabbit, goat, horse, andso on, is preferably used.

[0102] A polyclonal antibody can be obtained from serum of an animalsubjected to the above-described immunological means by a well-knownmethod for collecting antibodies. A preferable means is exemplified byimmunoaffinity chromatography.

[0103] A monoclonal antibody can be produced by collectingantibody-producing cells (for example, a lymphocyte derived from aspleen or a lymph node) from the animal subjected to the above-describedimmunological means, followed by introducing a well-known transformationmeans with a permanently proliferating cell (for example, myeloma strainsuch as P3/X63-Ag8 cells.) For example, the antibody-producing cells arefused with the permanent proliferating cells by a well-known method toprepare hybridomas. Then, the hybridomas are subjected to cloning,followed by selecting ones producing the antibody that recognizesspecifically the above-described polypeptide or peptide to collect theantibody from a culture solution of the hybridoma.

[0104] The polyclonal antibody or the monoclonal antibody thus obtained,which recognizes and binds to the above-described polypeptide orpeptide, can be utilized as an antibody for purification, a reagent, alabeling marker and so on.

[0105] (Screening and Compound Obtained by Screening)

[0106] The above-described polypeptide or peptide, the polynucleotideencoding the same and the complementary strand thereof, the celltransformed based on the information concerning the amino acid sequenceand nucleotide base sequence, or the antibody immunologicallyrecognizing the same provide an effective means for screening asubstance capable of inducing and/or activating CTL, when using themsolely or in combination with each other. The screening method can beconstructed utilizing a well-known screening system. For example, asshown in Examples herein, using a system in which the activation of CTLby the antigen-presenting cells that are pulsed with the tumor antigenpeptide, is measured on the basis of the amount of IFN-γ production fromCTL. Addition of a test substance to the system allows one to select thesubstance inducing and/or activating CTL and the substance enhancing theinduction and/or the activation. This system describes one screeningmethod, but the screening method according to the present invention isnot limited thereto.

[0107] A compound obtained by the above-described screening method isalso part of the present invention. The compound may be a compoundenhancing the recognition of the polypeptide or the peptide by CTLthrough an interaction with the polypeptide or the peptide according tothe present invention, and/or HLA-A2. Further, it may be a compoundenhancing the expression of the polynucleotide according to the presentinvention through an interaction with the polynucleotide. The compoundthus selected can be used in a pharmaceutical composition by selectingones having both biological usefulness and low toxicity.

[0108] (Pharmaceutical Composition)

[0109] The polypeptide or peptide according to the present invention canbe used for activating and/or inducing the HLA-A2-restrictedtumor-specific cytotoxic T lymphocytes, as a tumor antigen or a tumorantigen peptide. In other words, the method for inducing CTL, which ischaracterized in that the above-described polypeptide or peptide isused, and a inducer of CTL comprising the above-described polypeptide orpeptide are included in the scope of the present invention.

[0110] The polypeptide or the peptide according to the presentinvention, the polynucleotide encoding the polypeptide and thecomplementary strand thereof, the recombinant vector prepared based onthe information of their amino acid sequences and nucleotide basesequences, the cell transformed with the recombinant vector, or theantibody immunologically recognizing the polypeptide or the peptide, thecompound enhancing the recognition of the polypeptide or the peptide byCTL through interaction with the polypeptide or the peptide, and/orHLA-A2, or the compound enhancing expression of the polynucleotidethrough interaction therewith can used a pharmaceutical compositioncomprising at least one species thereof, when using them solely or incombination with each other.

[0111] Concretely, for example, composition consisting of thepolypeptide or the peptide according to the present invention, and thepharmaceutical composition comprising the polypeptide or the peptideaccording to the present invention can be used as a so-calledanti-cancer vaccine. In such a case, in order to activate thecell-mediated immunity, the polypeptide or the peptide according to thepresent invention can be used in the presence or absence of an adjuvantwith or without linking such to a carrier. Any carrier can be used aslong as it is not harmful to the human body. For example, cellulose, apolymerized amino acid, or albumin is exemplified, but the carrier isnot limited thereto. A dosage form is properly chosen from those towhich the well-known means for preparing a polypeptide or a peptide areapplied. The amount thereof to be administered depends on a degree ofrecognition of the peptide by CTL, and is generally 0.01 mg to 100mg/day/adult human body, preferably 0.1 mg to 10 mg/day/adult human bodyas an amount of active substance. Such an amount is administered onceevery several days or every several months.

[0112] Alternately, an effective action of an anti-cancer vaccine canalso be obtained by collecting a mononuclear cell fraction from theperipheral blood of a patient, and culturing the fraction with a peptideaccording to the present invention, followed by returning themononuclear cell fraction, in which CTL are induced and/or activated,back into the blood of the patient. Culture conditions, such as theconcentration of mononuclear cells and the concentration of thepolypeptide or the peptide according to the present invention when theyare cultured, can be readily determined. Further, a substance, such asinterleukin 2 (IL-2) having an ability to induce the growth oflymphocytes may be added to the culture.

[0113] In the case of using the polypeptide or the peptide according tothe present invention as an anti-cancer vaccine, using even only onepolypeptide or one peptide is effective as an anti-cancer vaccine.However, plural kinds of the above-described polypeptide or peptide canbe used in combination. CTL of the cancer patient is the population ofcells recognizing various tumor antigens, so that using plural kinds ofpolypeptides or peptides as an anti-cancer vaccine may give a highereffect than using only one kind.

[0114] The above-described composition, inducer of the cytotoxic Tlymphocytes, anti-cancer vaccine and pharmaceutical composition areuseful for treatment of a cancer disease such as pancreatic cancer,colon cancer, or stomach cancer.

[0115] In addition, the polynucleotide encoding the polypeptideaccording to the present invention and the complementary strand thereofare also useful for gene therapy of a cancer disease such as pancreaticcancer, colon cancer, or stomach cancer.

[0116] A method in which these polynucleotides are present in a vectorand directly introduced in vivo, and a method in which cells arecollected from a donor followed by introducing polynucleotides presentin a vector in vitro, can be both utilized. Retrovirus, adenovirus, andvaccinia virus exemplify the vectors, and retrovirus-related ones arepreferred. Needless to say, these viruses show deficiency forreplication. The amount of administration thereof can depend on thedegree of recognition by CTL of the polypeptide encoded by thepolynucleotide. Generally, as a DNA content encoding the tumor antigenpeptide according to the present invention, the amount ranges from 0.1μg to 100 mg/day/adult human body, preferably 1 μg to 50 mg/day/adulthuman body. This amount is administered once every several days or everyseveral months.

[0117] (Measuring Method for Diagnosis and Reagent)

[0118] The polypeptide or the peptide according to the presentinvention, the polynucleotide encoding the polypeptide and thecomplementary strand thereof, and the antibody immunologicallyrecognizing the polypeptide or the peptide can be used independently fora diagnostic marker and a reagent etc. The present invention alsoprovides a reagent kit comprising one or more containers in which one ormore species thereof are present. For the preparation thereof, it issufficient to use a well-known means for their preparation according toeach of polypeptide or peptide, polynucleotide, or antibody.

[0119] Diagnostic means for a disease related to expression or activityof the polypeptide or the peptide according to the present invention canbe carried out, for example, utilizing the interaction or reactivitywith the polynucleotide encoding the polypeptide, by determining theexisting amount of the corresponding nucleic acid molecule, and/ordetermining a distribution of the polypeptide or the peptide in anindividual living body, and/or determining a presence of the polypeptideor the peptide, and the existing amount in a sample derived from theindividual body. In other words, measurement is carried outquantitatively or qualitatively for the polypeptide or the peptideaccording to the present invention or the polynucleotide encoding thesame as the diagnostic marker. As a method for quantitative orqualitative measurement of the polypeptide or the peptide or the nucleicacid encoding the same, which are present in the sample, a well-knownmethod can be utilized. Radioimmunoassay, competitive binding assay,Western blotting analysis, ELISA, and the like exemplify such a method.In addition, the nucleic acid molecule can be detected and quantified atan RNA level by using, for example, amplification, polymerase chainreaction (PCR,) RT-PCR, RNase protection, Northern blotting method, andother hybridization methods.

[0120] The sample subjected to measurement is exemplified by the cellsderived from an individual human body present in for example, blood,urine, saliva, spinal fluid, tissue biopsy, or autopsy material, and thelike. The nucleic acid molecule subjected to measurement is obtainedfrom the each sample described above by a well-known method for nucleicacid preparation. For the nucleic acid molecule, genomic DNA can bedirectly used for detection, or it may be enzymatically amplified byusing PCR or any other amplification method before the analysis. RNA orcDNA may be similarly used. In comparing with a normal genotype, adeletion or insertion can be detected in accordance with a size changeof an amplification product. Hybridizing the amplified DNA with thelabeled DNA encoding the above-described polypeptide can identify pointmutations.

[0121] Detecting mutation of, reduction of, and increase in thepolypeptide according to the present invention and the DNA encoding thepolypeptide by the above-described measuring method, makes it possibleto diagnose diseases, to which the polypeptide is associated, such aspancreatic cancer, colon cancer, or stomach cancer.

EXAMPLES

[0122] The present invention will be illustrated more concretely withthe following examples, but is not limited thereto.

Example 1

[0123] (Establishment of HLA-A2-Restricted CTL)

[0124] The HLA-A2-restricted tumor-specific cytotoxic T lymphocyte linewas established from tumor infiltrating lymphocytes (TIL) of a colontumor patient (HLA-A0207/3101, HLA-B46/51, HLA-Cw1) (Int. J. CANCER,81:459-466, 1999; J. Immunol., 163:4994-5004, 1999.) Specifically, TILobtained from the colon tumor patient was cultured for a long period upto 50 days or longer by adding 100 U/ml of recombinant human interleukin2 (IL-2.) Every 7 days, a portion of TIL activated by IL-2 was collectedand cultured together with various kinds of tumor cells or normal cellsto assay its CTL activity by measuring the produced IFN-γ and bymeasuring ⁵¹Cr released from the cancer cells (J. Immunol.,163:4994-5004, 1999.) IFN-γ was measured by an enzyme-linkedimmunosorbent assay (ELISA). At day 58 after the start of the culture,OK-CTLp, which is one of sublines showing HLA-A2-restrictedtumor-specific CTL activity, was obtained. OK-CTLp obtained is a cellpopulation in which 80% of the cells have a phenotype of CD3⁺CD4⁻CD8⁺and 20% of the cells have a phenotype of CD3⁺CD4⁺CD8⁻. Using OK-CTLp asan effector cell and culturing it together with various kinds of cellsthat are used as a target cell, such as a tumor cell, the cytotoxicityto the target cell and activation of OK-CTLp were measured using the⁵¹Cr-release test and by using IFN-γ production as an indicator,respectively. The results are each presented in FIG. 1 and FIG. 2.

[0125] OK-CTLp obtained, as shown in FIGS. 1 and 2, recognizedHLA-A0201⁺Panc-1 pancreatic adenocarcinoma cell, SW620 colonadenocarcinoma cell, HLA-A0206⁺KE3 esophageal squamous-cell carcinoma(SCC) cell and HLA-A0207⁺CA9-22 oral SCC cell to produce IFN-γ andrepresent sufficient cytotoxicity. However, no cytotoxicity was shownagainst HLA-A2⁻ tumor cells, such as QG56 lung adenocarcinoma cell,RERF-LC-MC lung adenocarcinoma cell, and COLO320 colon adenocarcinomacell, and autologous Epstein-Barr virus transformed B cell (EBV-B) andautologous phytohemagglutinin (PHA)-blastoid T cells both derived fromthe normal cells. Further, OK-CTLp lysed all of the HLA-A2⁺ tumor cellstested (R27 breast cancer cell, HAK-2 primary hepatocellular carcinomacell, SK-MEL-5 melanoma cell, and SF126 astrocytoma cell, which areHLA-A0201⁺, and HLA-A0206⁺PC9 lung adenocarcinoma cell, and 1-87 lungadenocarcinoma cell and OMC-4 cervical SCC cell, which are HLA-A0207⁺.)The CTL activity was inhibited by an anti-HLA class I monoclonalantibody (mAb), an anti-CD8 mAb or an anti-HLA-A2 mAb, but not inhibitedby other mAbs (FIG. 2.) This result revealed that OK-CTLp recognizes theabove-described tumor cells in a HLA-A2-restricted manner and showscytotoxicity.

[0126] Meanwhile, the genotype of the HLA class I alleles of theabove-described tumor cells has been disclosed (J. Immunol.,163:4994-5004, 1999.) The serotype of the HLA class I of theabove-described patients was determined by applying a conventionalmethod using the peripheral blood mononuclear cells (PBMC). In addition,the HLA-A2 subtype was determined by a sequence-specific oligonucleotideprobe method and direct DNA sequencing. The phenotype of OK-CTLp wasanalyzed by direct immunofluorescence analysis using anti-CD3 mAb,anti-CD4 mAb, or anti-CD8 mAb (made by Nichirei) or anti-TCRαβ mAb(WT31, Becton Dickinson), which were labeled with fluoresceinisothiocyanate (FITC). In addition, the antibodies used to analyze forHLA-A2 restriction and specificity of OK-CTLp were anti-HLA class I mAb(W6/32, IgG2a,) anti-HLA-A2 mAb (BB7.2, IgG 2b,) anti-CD8 mAb (Nu-Ts/c,IgG2a,) anti-HLA class II mAb (H-DR-1, IgG2A,) and anti-CD4 mAb(Nu-Th/i, IgG1.) Anti-CD13 mAb (MCS-2, IgG2a) and Anti-CD14 mAb(JML-H14, IgG1) were used as an isotype-matching control mAb.

Example 2

[0127] (Isolation and Identification of cDNA Clone Encoding TumorAntigen)

[0128] A gene encoding the tumor antigen of the Panc-1 tumor cellrecognized by OK-CTLp was isolated and identified according to thewell-known gene expression cloning method (J. Immunol., 163:4994-5004,1999.) Specifically, poly(A)⁺ RNA of the Panc-1 tumor cells wasconverted to CDNA, and ligated with a SalI adapter so as to insert intothe expression vector pCMV-SPORT-2 (Invitrogen Corp.)

[0129] cDNAs of HLA-A0207, HLA-A2402, or HLA-A2601 were obtained byreverse transcriptase polymerase chain reaction (RT-PCR) and cloned intothe eukaryote expression vector pCR3 (Invitrogen Corp.)

[0130] 200 ng of the above-described plasmid DNA pool or clones ofPanc-1 cell cDNA library was mixed with 200 ng of the cDNA of HLA-A0207in 100 μl of Opti-MEM (Invitrogen Corp.) for 30 min. 50 μl of thismixture was added to COS-7 cells (5×10³) and incubated for 6 h in a96-well U-bottom type microculture plate (Nunc Corp.) forco-tranduction. Then, RPMI-1640 culture medium containing 10% FCS wasadded to and culturing was carried out for 2 days, followed by theaddition of OK-CTLp (5×10⁴) to each well. After a further 18 hincubation, 100 μl of the supernatant was collected and IFN-γ productionwas measured thereon by ELISA. In this case, using COS-7 cells to whichthe gene had not been transfected as a target, IFN-γ production byOK-CTLp was examined and the value of IFN-γ produced was subtracted as abackground from that of each measurement. As a result, seven cDNA cloneswere obtained, which enhanced IFN-γ production by OK-CTLp throughrecognition by OK-CTLp.

[0131] The nucleotide sequence of the seven cDNA clones obtained wasdetermined by dideoxynucleotide sequencing method using a DNA sequencingkit (Perkin Elmer Corp.) and using an ABI PRISM™377DNA sequencer (PerkinElmer Corp.) In addition, the amino acid sequence encoded by each clonewas deduced from the nucleotide base sequence. Also, a homology searchof the nucleotide base sequence of these clones was conducted throughaccessing GenBank. The results are presented in the above-describedTable 1. With regard to clone 3 among the seven cDNA clones (clones 1 to7) obtained, the sequence of an initial clone, which was obtained by thegene expression cloning method described above, was 25 bp shorter at the5′-terminal region than that of WHSC2 showing high homology, so thatfull-length cDNA was obtained from the cDNA library of the Panc-1 cellby a standard colony hybridization method using the clone labeled with³²P as a probe.

[0132] As shown in FIGS. 3A and 3B, clones 1 to 7 were each recognizedby OK-CTLp to enhance IFN-γ production of OK-CTLp. However, OK-CTLp didnot recognize COS-7 cells to which HLA-A0207 cDNA and the cDNA cloneused as a negative control were cotransfected, or COS-7 cells to whichany one of cDNA clones 1 to 7 and the cDNA of HLA-A2402 or HLA-A2601were cotransfected, and did not show the IFN-γ production. When variousconcentrations of cDNA clones 1 to 6 were cotransfected into COS-7 cellstogether with 100 ng of HLA-A0207 cDNA or HLA-A2402 cDNA, IFN-γproduction by OK-CTL was observed in a dose-dependent manner (FIGS. 4Ato 4F.)

[0133] Expression of the mRNA of these genes was examined by Northernblotting analysis. The same expression pattern was observed except forgene 5. These genes are expressed commonly in the tumor cells and normalcells. However, expression levels in tumor cells such as Panc-1 cell,SW620 cell, and CA9-22 cell were significantly higher than that innormal cells, such as the T cell stimulated by PHA and a B celltransformed by Epstein-Barr virus (EBV-B). Expression of mRNA of gene 5was barely detected under these experimental conditions. The reason maybe that expression of gene 5 is rare as proven by the fact that colonyhybridization using clone 5 labeled with ³²P gives only 3 clones from anabout 1×10⁶ CDNA library.

Example 3

[0134] (Establishing OK-CTL Clone)

[0135] Since CTL activated by recognizing the tumor antigen is apopulation of cells recognizing plural kinds of tumor antigens, theabove-described OK-CTLp was subjected to cloning by limiting dilutionculture (0.3, 0.5, 1, 2, and 4 cells/well) to obtain an OK-CTL clone (J.Immunol., 163:4994-5004, 1999.) These clones are those having CTLactivity selected by culturing them together with COS-7 cells into which100 ng/well of any one of the above-described seven cDNA clones and 100ng/well of HLA-A0207 cDNA were cotransfected, or with the tumor cells,in a cell ratio of 1:1, and measuring IFN-γ production. Specifically,three hundred CTL clones were obtained from the parent line OK-CTLp by alimiting dilution culture. Eighty CTL clones among them hadHLA-A2-restricted tumor-specific CTL activity and expressed thephenotype of CD3⁺ CD4⁻ CD8⁺ and TCR αβ⁺. Among them, 2, 3, 1, 3, 2, and4 CTL clones showed reactivity to the COS-7 cells expressing clone 1,clone2, clone 3, clone 4, clone 5, and clone 6, respectively. In otherwords, it was revealed that the tumor antigen recognized by CTL differsin accordance with the CTL clones. Table 4 shows data of fifteen typicalCTL clones. This suggested that OK-CTLp, i.e., CTL derived from thecancer patient, is a population of cells recognizing plural kinds oftumor antigens. TABLE 4 cDNA expressed in COS-7 cell together withHLA-A0207 cDNA clone clone clone clone Clone clone 1 2 3 4 5 6 no CTLclone (UBE2V) (HNRPL) (WHSC2) (EIF4EBP1) (ppMAPkkk) (2-5 OAS3) cDNAPanc-1   2-2-H3 110 0 0 0 0 0 0 340   2-1-H12 134 0 0 0 0 0 0 264  1-2-D7 0 200  0 0 0 0 0 235   1-2-D12 0 >1000     0 0 0 0 0 >1000  4-1-H8 0 133  0 0 0 0 0 84   4-2-A11 0 0 100  0 0 0 0 725 0.5-1-H12 00 0 >1000     0 0 0 >1000 0.5-1-D6 19 0 0 118  0 13  0 448   4-2-B3 0 00 95  0 0 0 100   2-1-F4 0 0 0 0 >1000     0 0 >1000 0.5-1-H2 0 0 0 081  0 0 122 0.5-1-D7 27 34  0 0 0 110  0 304 0.5-2-A4 0 0 0 0 0 113 0 >1000   1-2-D1 21 0 22 44  0 61  0 78   2-2-B4 0 0 0 0 0 >1000     0>1000

Example 4

[0136] (Preparation of Tumor Antigen Peptide and its CTL-InducingActivity)

[0137] In order to obtain the tumor antigen peptide derived from theseven tumor antigen genes, which were obtained in Example 2 and caninduce CTL in a HLA-A2-restricted manner, a peptide having an HLA-A2binding motif (a specific sequence) was searched for in the literature(J. Immunol., 152:163, 1994; Immunogenetics, 41:178, 1994,) and peptidesof 9-mer to 11-mer, which were different from each other and suited tothe motif obtained, was designed and synthesized based on the amino acidsequence encoded by the above-described genes 1 to 7 and the amino acidsequence of UBE2V, HNRPL, WHSC2, EIF4EBP1, ppMAPkkk, 2-5 OAS3, and CPSFhaving high homology with these genes. The purity of the peptidesobtained was each 70% or higher.

[0138] Binding activity of the peptide to the HLA-A0201 molecule wastested using a T2 cell mutant strain (Cancer Res., 54:1071-1076, 1994.)The T2 cell expresses the HLA-A2 molecule on a cell surface withoutbinding to a peptide, because of deficiency of TAP. Specifically, thesynthesized peptide (10 μM) and the T2 cells were incubated at 26° C.for 3 h and, subsequently, incubated in 5% CO₂ and 95% air at 37° C. for3 h. Thus, T2 cells, on whose surface the peptide was presented byHLA-A2, were obtained. The cells were incubated together withanti-HLA-A2 mAb (BB7.2) followed by staining with R-phycoerythrin linkedF (ab′)₂ rabbit anti-mouse immunoglobulin (Ig) (DAKO Corp.) Then, theexpression pattern was analyzed by employing FACScan (Beckman DickinsonCorp.), which resulted in confirmation that HLA-A0201 molecules with thepeptide were expressed on the cell surface.

[0139] In order to test for recognition of a peptide by CTL, the T2cells previously pulsed with each peptide (10 μM) was used as a targetcell (T), and OK-CTLp or OK-CTL clone was used as an effector cell (E).The target cell and the effector cell were incubated for 18 h, thesupernatant collected, followed by measuring IFN-γ contained in thesupernatant by an ELISA. In the case where OK-CTLp was used as theeffector, an E/T ratio was set to 10:1. In the case where the OK-CTLclone was used, it was set to 2:1 to conduct the test. On the otherhand, in case where the OK-CTL clone was used to test for the CTLactivating ability of the peptide, the clone was used which recognizedthe gene product encoding the peptide being examined. Using IFN-γproduction of OK-CTLp or OK-CTL clone against the T2 cells, which hadnot been pulsed with the peptide, as a background, subtraction wasperformed from each measurement value. The results were shown in FIGS. 5to 10. FIG. 5, FIG. 6, FIG. 7, FIG. 8, FIG. 9, and FIG. 10 each show theresult of the peptide derived from gene 1 and UBE2V, gene 2 and HNRPL,gene 3 and WHSC2, gene 4 and EIF4EBP1, and gene 5 and ppMAPkkk, and gene6 and 2-5 OAS3.

[0140] Moreover, the various concentrations of each peptide were usedfor incubation together with the T2 cells to examine the CTLclone-activating ability, resulting in finding that the CTL clone can beactivated by each peptide in a dose-dependent manner. Representativeexamples of peptides derived from gene 1 and UBE2V, gene 2 and HNRPL,gene 3 and WHSC2, gene 4 and EIF4EBP1, gene 5 and ppMAPkkk, and gene 6and 2-5 OAS3 were presented in FIGS. 11A-11F, respectively. On the otherhand, Table 5 shows peptides derived from gene 7 and CPSF. In Table 5,the peptide derived from EBV and the peptide derived from influenzavirus are positive controls, which can activate CTL.

[0141] As the result of these experiments, it was revealed that thepeptides shown in Table 2 described-above can activate OK-CTLp and/orOK-CTL clone to produce IFN-γ. TABLE 5 IFN-γ production (pg/ml)Concentration of peptide Peptide 0.1 μM 1 μM 10 μM 50 μM Derived 0  93693  35 from influenza virus Derived 119 390 371 117 from EBV P20 344643  0  34 P21 428 501  81 195 P22 254 1027  431 407 P23 6360  0 345 160P24 728 Detection 103  2 limit or higher P25 69 1569   25 122 P26 141418 1239   0 P27 352 889 250  0 P28 898 Detection  0 144 limit or higherP29 0 Detection  86 362 limit or higher P30 88 7001  Detection Detectionlimit or higher limit or higher P31 443 Detection Detection 314 limit orhigher limit or higher

Example 5

[0142] (CTL Induction from Peripheral Blood Mononuclear Cells Derivedfrom Cancer Patient by a Peptide)

[0143] Among the peptides that were able to enhance IFN-γ productionfrom the OK-CTLp or OK-CTL clone in Example 4, those from P1 to P19 wereexamined for their ability to induce CTL from peripheral bloodmononuclear cells derived from a cancer patient. The method for inducingCTL by a peptide was according to the well-known method (J. Exp. Med.,187:277-288, 1998; Cancer Res., 59:4056-4063, 1999.) Specifically,autologous peripheral blood mononuclear cells (PBMC) derived from acancer patient, from whom OK-CTLp was obtained, were incubated togetherwith the peptide (10 μM.) The cells were re-stimulated at day 10 and 14after the start of culturing, using autologous PBMCs as an antigenpresenting cell (APC) which were pulsed with 10 μM of the same peptidefor 2 h and exposed to irradiation (30 gray.) At day 21 after the startof culturing, the cells were collected to test for surface phenotype. Inaddition, the cells were examined for the recognition of various targetcells, using IFN-γ production measured by ELISA when they were culturedtogether with the target cells as an indicator. As a target cells, SW620cell, CA9-22 cell, and Panc-1 cell, which are the HLA-A2⁺ tumor cells,were used. The result was shown in Table 6. TABLE 6 IFN-γ production(pg/ml) from peripheral blood mononuclear cells in recognition ofvarious target cells QG56 RERF-LC-MS COLO320 SW620 CA9-22 Panc-1 PeptideMFI (HLA-A26/26) (HLA-A11/11) (HLA-A24/24) (HLA-A0201/24) (HLA-A0207/24)(HLA-A0201/11) P1 571 0 26 50 235 81 492 P2 607 0 0 5 53 0 0 P3 910 0 044 188 58 289 P4 1008 0 0 0 60 0 0 P5 637 0 0 38 500 96 638 P6 819 0 840 344 863 527 P7 783 0 0 0 344 0 54 P8 499 0 26 0 142 165 186 P9 832 027 0 194 98 339 P10 504 0 0 0 108 130 163 P11 1089 0 0 0 893 62 >1000P12 780 0 0 40 46 0 197 P13 656 0 15 0 151 95 115 P14 591 0 0 0 112 184265 P15 789 0 32 0 199 219 502 P16 887 0 0 0 0 147 113 P17 660 0 00 >1000 >1000 691 P18 657 29 0 0 >1000 70 >1000 P19 775 0 30 66 55 48105 no 491 0 0 36 0 0 17 Peptide

[0144] PBMC stimulated in vitro using P1, P3, P5, P6, P8, P9, P10, P11,P13, P14, P15, P17, or P18 among the 19 peptides recognized SW620 cells,CA9-22 cells and Panc-1 cells, which are the HLA-A2⁺ tumor cells,produced IFN-γ in a significant amount. However, the PBMC barelyrecognized the HLA-A2⁻ tumor cell. On the other hand, P2, P4, P7, P12,and P16 also induced CTL recognizing any one of the HLA-A2⁺ tumor cells.PBMC stimulated by P19 recognized not only the tumor cells expressingHLA-A2, but also the tumor cells expressing other types of HLA. IFN-γproduction from these CTL was inhibited by anti-HLA class I mAb,anti-CD8 mAb, or anti-HLA-A2 mAb, and not inhibited by other mAbs. Inaddition, when peripheral blood mononuclear cells (PBMC) were preparedfrom blood of two HLA-A0201⁺ patients (a colon cancer patient and apancreatic cancer patient) to examine CTL induction by the peptides, thesame result as above was obtained. In other words, it was found that theabove-described peptides can induce HLA-A2-restricted CTL from theperipheral blood mononuclear cells of the patient.

[0145] Binding affinity of the peptides to HLA-A0201 molecule wasexpressed as a relative mean fluorescence intensity (MFI) of the HLA-A2molecule. The MFI of positive and negative controls were 898 and 490,respectively. It can be supposed that binding affinity of a peptide tothe HLA-A0201 molecule has no correlation with induction of CTL by thepeptide.

[0146] Moreover, CTL-activating ability of these 19 peptides wasdirectly examined in a ⁵¹Cr-releasing test using toxicity to the targetcells as an indicator. Specifically, the above-described PBMC, in whichCTL was induced, was further recultured to proliferate in the presenceof autologous APC, IL-2, and a corresponding peptide. At about day 21 to28 after the start of reculturing, PBMC was collected and cytotoxicitythereof was tested again by measuring IFN-γ and by a 6-h ⁵¹Cr-releasingtest. The results were shown in FIGS. 12 to 17. PBMC of the cancerpatient, which was stimulated by these peptides, lysed the HLA-A2⁺ tumorcells. However, the autologous EBV-B cells and the T cells stimulated byPHA, both of which were derived from normal cells and were expressingHLA-A2, and the HLA-A2⁻ tumor cell RERF-LC-MS, were not lysed. However,PBMC stimulated by peptides P14, P15, and P17 also showed cytotoxicityto the autologous EBV-B cells. Moreover, PBMC stimulated by P19 showedhigh cytotoxicity to the autologous EBV-B cells. In addition, PBMCstimulated by these peptides showed cytotoxicity to the T2 cells, whichwas pulsed with the same peptide as that used for stimulation of PBMC,in a dose dependent manner. Typical examples are shown in FIGS. 18A-18E.From these above, it was found that the above-described peptides caninduce CTL, which shows HLA-A2-restricted cytotoxicity, from theperipheral blood mononuclear cells of the cancer patient.

Example 6

[0147] (CTL Induction from Peripheral Blood Mononuclear Cell of Patientby Peptide)

[0148] For six peptides (P21, P22, P24, P26, P30, and P32) with a purityof 95% or higher among peptides from P20 to P32 derived from gene 7 andCPSF among the tumor antigen peptide obtained in Example 4, the activityto induce CTL in vitro from human peripheral blood mononuclear cells(PBMC) was examined using IFN-γ production as an indicator. PBMC usedwas prepared from each peripheral blood of sixteen HLA-A2-positivecancer patients (4 patients with pancreatic cancer, seven patients withstomach cancer, and 5 patients with colon cancer) and six healthyindividuals. Specifically, 1×10⁵ PBMC was added to each well of 96-wellU-bottom type microculture plate (Nunc Corp.) and incubated togetherwith 10 μM of each of the above-described peptides in 200 μl culturemedium. The culture medium consisted of 45% RPMI-1640, 45% AIM-V(Invitrogen Corp.,) 10% fetal calf serum (FCS), 100U/ml of humaninterleukin-2, and 0.1 μM MEM nonessential amino acid solution(Invitrogen Corp.). At day 4 and day 7 after the start of culturing,half of the culture medium was removed and replaced with theabove-described composition containing each corresponding peptide. Atday 10 after the start of culturing, the cells were collected andwashed, followed by reacting with T2 cells, which were pulsed with eachcorresponding peptide, to measure the amount of IFN-γ produced.

[0149] Meanwhile, the above-described cells cultured for 10 days afterstimulation by the peptide were further cultured for 10 days. Thecytotoxicity of the obtained cells against the Panc-1 pancreaticadenocarcinoma cell (HLA-A2) was measured by the standard 6-h⁵¹Cr-releasing test in an E/T ratio of 10:1. The results obtained arepresented as % specific lysis (table 7). Together with this step,cytotoxicity against SSTW-9 tumor cell as the HLA-A2⁻ tumor cell wasmeasured to use as a background that was subtracted from theabove-described result.

[0150] As a result, induction of HLA-A2-restricted CTL from PBMC by P21,P22, P23, P26, P30, and P32, which was specific to each peptide, wasobserved in patients of 31% ({fraction (5/16)},) 38% ({fraction(6/16)},) 25% ({fraction (4/6)},) 31% ({fraction (5/16)},) 44%({fraction (7/16)},) and 7% ({fraction (1/16)}) of the above-describedsixteen patients, respectively. On the other hand, induction of CTL byP21 and P22 from the PBMC of healthy individuals was found in 50%({fraction (3/6)}) and 33% ({fraction (2/6)},) respectively. However,the other peptides did not induce CTL from the PBMC of healthyindividuals (Table 7). The above-described CTL induced from PBMC of thecancer patients by using the peptide showed cytotoxicity against Panc-1pancreatic adenocarcinoma cell, and also SW620 colon adenocarcinoma cell(HLA-A2/A24) and KWS stomach adenocarcinoma cell (HLA-A2), both of whichare the HLA-A2⁺ tumor cells. However, lysis was not observed in SSTW9stomach adenocarcinoma cell (HLA-A24), which is the HLA-A2⁻ tumor cell,or the PHA-blastoid T cells or the EBV-B transformed B cells, both ofwhich express HLA-A2 and are not the tumor cells. Recognition of thetumor cell by the above-described CTL was inhibited by anti-HLA class ImAb, anti-CD8 mAb, or anti-HLA -A2 mAb, and not inhibited by other mAb.TABLE 7 INF-γproduction (pg/ml)/% specific lysis Origin of PBMC P21 P22P23 P26 P30 P32 pancreatic 1 0 0 0 0 0 0 cancer 2 7 38/37 41/53 35/36 1212 patient 3 0 0 0 0 0 0 4 543/12  8 72/10 77/20 73/11 0 stomach 5 0 0 053/11 0 0 cancer 6 0 0 0 0 0 0 patient 7 0 0 46/10 0 0 0 8 5 0 0 11146/18  0 9 1500</ 0 0 8 92 0 27 10 0 0 0 0 46/26 0 11 136/18  0 0 0 0 0colon cancer l2 0 92/28 71/18 0 0 57/18 patient 13 0 179 138/10  0 41/120 14 66/11 80/19 48 0 33/10 0 15 0 5 14 40/12 0 0 16 140 0 0 45 68 4healthy HD1 0 0 0 0 0 0 individuals HD2 0 0 13 0 0 0 HD3 58 0 8 0 14 0HD4 5 0 6 0 0 0 HD5 224 0 83 13 0 0 HD6 97 14 45 0 0 0

Example 7

[0151] (Isolation and Identification of cDNA Encoding Tumor Antigen)

[0152] From the genes encoding the tumor antigens (Biochim. Biophys.Res. Commun., 281:936-944, 2001), which were detected by the SEREX(Serological Analysis of Recombinant cDNA Expression Libraries) method(Proc. Natl Acad. Sci. USA, 92:11910-11813, 1995) and already reported,two tumor antigen genes, KM-PA-2 and KM-PA-4, which can activateHLA-A2-restricted CTL, were found by using the same method as that ofExample2. Specifically, cDNA clones each encoding KM-PA-2, KM-PA-4,KM-PA-5, KM-PA-14, KM-PA-15, or KM-PA-18 were packaged in pBluescriptvector, and digested by EcoRI and XhoI to insert into pCMV-SPORT2. ThesecDNAs in various concentrations were each coexpressed in COS-7 cellstogether with HLA-A0207 or HLA-A2402. Using the COS-7 cells as a targetcell, incubation was performed together with OK-CTLp. As a result, COS-7cells, to which KM-PA-2 or KM-PA-4 was cotransfected with HLA-A0207,induced IFN-γ production from OK-CTLp, in a dose dependent manner of thegene (FIGS. 19A-19B.) The SEREX method is a method for detection of thetumor antigen. However, among 1500 or more kinds of tumor antigensdetected by this method, those identified as tumor antigens capable ofinducing both cell-mediated immunity and humoral immunity are onlyMAGE-1, tyrosinase, and NY-ESO-1. Therefore, even a gene identified bythe SEREX method as encoding the tumor antigen cannot always activateCTL. It was first found that the above-described tumor antigen genes,KM-PA-2 and KM-PA-4, can activate CTL in an HLA-A2-restricted manner.

Example 8

[0153] (Preparation of Tumor Antigen and Activity for Inducing CTL fromPBMC of Cancer Patient)

[0154] In order to obtain the tumor antigen peptide derived from thetumor antigen genes, KM-PA-2 and KM-PA-4, obtained from Example 7,different peptides of 9-mer or 10-mer were designed based on the aminoacid sequences encoded by KM-PA-2 and KM-PA-4 and synthesized by awell-known method, in the same way as that in Example 4.

[0155] The ability of the synthesized peptide to induce CTL from theperipheral blood mononuclear cells of the colon cancer patient, fromwhom OK-CTLp was obtained, was examined in the same way as that inExample 6. As a result, as shown in FIGS. 20A-20B, PBMC that wasstimulated in vitro using any one of peptides P33 to P41 (SEQ ID NO: 33to SEQ ID NO: 41) derived from KM-PA-2 and peptides P42 to P44 (SEQ IDNO: 42 to SEQ ID NO: 44) derived from KM-PA-4, produced IFN-γ throughrecognition of the T2 cells (left-hand figure of FIGS. 20A-20B,) whichwere pulsed with the peptide corresponding to that used for stimulationof the PBMC, and the Panc-1 cell (right-hand of FIGS. 20A-20B,) which isthe HLA-A2⁺ tumor cell. However, the PBMC reacted merely to the HLA-A2⁻tumor cell. As a result, it was revealed that any one of theabove-described twelve peptides can induce the antigen specific CTL fromPBMC of the cancer patient in an HLA-A2-restricted manner and that theinduced CTL can recognize the above-described peptides to produce IFN-γin an HLA-A2-restricted manner. In addition, cytotoxicity of these CTLsinduced by the peptides was directly confirmed by the ⁵¹Cr-releasingtest in the same way as that in Example 6. FIGS. 21A-21C show arepresentative example of the result. As shown in FIGS. 21A-21C, CTLsinduced from PBMC of the cancer patient by P35, P39, or P40 lysed Panc-1cells and YPK-3 cells, both of which are HLA-A2⁺ tumor cells. However,the HLA-A2⁻ tumor cell PaCa-2, the EBV-B cell line OKAB2, and the Tcells stimulated by PHA, was not lysed. In other words, it was revealedthat the above-described peptides can induce CTL showing cytotoxicityfrom the peripheral blood mononuclear cells of the cancer patient in anHLA-A2-restricted manner. Further, CTL was also induced by theabove-described peptides from PBMC obtained from the cancer patient,such as a pancreatic cancer patient, in addition to the colon cancerpatient from whom OK-CTLp was obtained.

Example 9

[0156] In order to determine the phenotype of TCR expressed on the cellsurface of the CTL clone recognizing the above-described peptide, totalRNA of each clone was obtained from each 5×10⁶ CTL clones, which wereobtained in Example 3, using RNAzol™B (TEL-TEST Corp.) cDNA was preparedusing the Superscript™ Preamplification System (Invitrogen Corp.) forfirst strand cDNA synthesis. Single stranded cDNA was amplified bypolymerase chain reaction (PCR) using one of the 22 different Vβ primers(Vβ1 to 20) and 3′C β primers. PCR was performed for 35 cycles, wherein1 cycle comprised denaturation at 94° C. for 1 min, annealing at 58° C.for 2 min, and extension at 72° C. for 3 min. The PCR product wasinserted into plasmid pCR2 followed by transformation into Escherichiacoli using the TA cloning system (Invitrogen Corp.), selection ofcolonies, and plasmid preparation for determining the cDNA sequence.

[0157] As the result, two each of CTL clones reacting with the peptidesderived from UBE2V and gene 1, the peptide derived from HNRPL and gene2, and the peptide derived from 2-5 OAS3 and gene 6, expressed TCR-Vβ8.1, TCR-Vβ 3.2, and TCR-Vβ 14, respectively. In addition, CTL clonesrecognizing the peptide derived from WHSC2 and gene 3, the peptidederived from EIF4EBP1 and gene 4, the peptide derived from ppMAPkkk andgene 5, expressed TCR-Vβ 13.1, TCR-Vβ 8.1, and TCR-Vβ 18, respectively(Table 8-1 and Table 8-2).

[0158] Two each of CTL clones recognizing the peptide derived from UBE2Vand gene 1, the peptide derived from HNRPL and gene 2, and the peptidederived from 2-5 OAS3 and gene 6, expressed TCR possessing differentcomplementarity-determining regions 3 (CDR3) (an element responsible forbinding to the antigenic epitopes on the groove of the HLA class Imolecules), respectively. CTL clones recognizing the peptide derivedfrom WHSC2 and gene 3, the peptide derived from EIF4EBP1 and gene 4, thepeptide derived from ppMAPkkk and gene 5, expressed TCR possessingdifferent CDR3, respectively. The amino acid sequence of each CDR3 isthat shown with an underline in Table 8-2. TABLE 8-1 CTL clone EpitopesVβ Dβ Jβ Cβ 2-2-H3 UBE2V 43-51 8.1 2.1 2.3 2 2-1-H12 UBE2V 43-51 8.1 2.12.3 2 1-2-D7 HNRPL 140-148 3.2 1.1 2.7 2 1-2-D12 HNRPL 140-148 3.2 1.12.7 2 4-2-A11 WHSC2 103-111 13.1 2.1 2.7 2 4-2-B3 EIF4EBP1 51-59 8.1 1.11.1 1 0.5-1-H2 ppMAPkkk 432-440 18 1.1 1.1 1 1-2-D1 2-5 OAS3 666-674 142.1 2.3 2 2-2-B4 2-5 OAS3 666-674 14 2.1 2.3 2

[0159] TABLE 8-2 CTL clone Vβ Dβ* Cβ 2-2-H3 IYFNNNVPIDDSGMPEDRFSAKMPNASSLGLAGGEQFFGPGTRLTVL EDLKNVFPPE FSTLKIQPSEPRDSAVYFCAS 2-1-H12IYFNNNVPIDDSGMPEDRFSAKMPNAS SLGLAGGEQFFGPGTRLTVL EDLKNVFPPEFSTLKIQPSEPRDSAVYFCAS 1-2-D7 VSREKKERFSLILESASTNQTSMYLCASLDRSYEQYFGPGTRLTVT EDLKNVFPPE S 1-2-D12 VSREKKERFSLILESASTNQTSMYLCASLDRSYEQYFGPGTRLTVT EDLKNVFPPE S 4-2-A11 QGEVPNGYNVSRSTTEDFPLRLLSAAPSYGGGSSYEQYFGPGTRLTV EDLKNVFPPE SQTSVYFCAS T 4-2-B3IYFNNNVPIDDSGMPEDRFSAKMPNAS SRVSGEAFFGQGTRLTVV EDLKNVFPPEFSTLKIQPSEPRDSAVYFCAS 0.5-1-H2 DESGMPKERFSAEFPKEGPSILRIQQVSPTELDTEAFFGQGTRLTVV EDLKNVFPPE VRGDSAAYFCAS 1-2-D1VSRKEKRNFPLILESPSPNQTSLYFCA GGSTDTQYFGPGTRLTVL EDLKNVFPPE S 2-2-B4VSRKEKRNFPLILESPSPNQTSLYFCA GGSTDTQYFGPGTRLTVL EDLKNVFPPE S

[0160] Industrial Applicability

[0161] According to the present invention, HLA-A2-restricted cytotoxic Tlymphocytes can be induced, which makes it possible to achieve aspecific immunotherapy for pancreatic cancer, colon cancer, and stomachcancer. HLA-A2 alleles are found in 23% of African Blacks, 53% ofChinese, 40% of Japanese, 49% of Northern Caucasians, and 38% ofSouthern Caucasians. Consequently, the present invention can be expectedits great contribution to cancer therapy. Moreover, the presentinvention greatly contributes to fundamental research on the moleculerelated to recognition by T cells of a pancreatic cancer cell, a coloncancer cell, stomach cancer, and so on.

1 197 1 9 PRT Artificial Designed peptide having an ability to activateHLA-A2 restricted Cytotoxic T lymphocytes 1 Arg Leu Gln Glu Trp Cys SerVal Ile 1 5 2 10 PRT Artificial Designed peptide having an ability toactivate HLA-A2 restricted Cytotoxic T lymphocytes 2 Leu Leu Leu Leu AlaArg Trp Glu Asp Thr 1 5 10 3 9 PRT Artificial Designed peptide having anability to activate HLA-A2 restricted Cytotoxic T lymphocytes 3 Leu IleAla Asp Phe Leu Ser Gly Leu 1 5 4 9 PRT Artificial Designed peptidehaving an ability to activate HLA-A2 restricted Cytotoxic T lymphocytes4 Leu Leu Gln Asp Trp His Val Ile Leu 1 5 5 9 PRT Artificial Designedpeptide having an ability to activate HLA-A2 restricted Cytotoxic Tlymphocytes 5 Ile Leu Pro Arg Lys His His Arg Ile 1 5 6 9 PRT ArtificialDesigned peptide having an ability to activate HLA-A2 restrictedCytotoxic T lymphocytes 6 Ala Leu Val Glu Phe Glu Asp Val Leu 1 5 7 9PRT Artificial Designed peptide having an ability to activate HLA-A2restricted Cytotoxic T lymphocytes 7 Cys Leu Tyr Gly Asn Val Glu Lys Val1 5 8 9 PRT Artificial Designed peptide having an ability to activateHLA-A2 restricted Cytotoxic T lymphocytes 8 Phe Met Phe Gly Gln Lys LeuAsn Val 1 5 9 10 PRT Artificial Designed peptide having an ability toactivate HLA-A2 restricted Cytotoxic T lymphocytes 9 Asn Val Leu His PhePhe Asn Ala Pro Leu 1 5 10 10 9 PRT Artificial Designed peptide havingan ability to activate HLA-A2 restricted Cytotoxic T lymphocytes 10 AlaSer Leu Asp Ser Asp Pro Trp Val 1 5 11 9 PRT Artificial Designed peptidehaving an ability to activate HLA-A2 restricted Cytotoxic T lymphocytes11 Ile Leu Gly Glu Leu Arg Glu Lys Val 1 5 12 9 PRT Artificial Designedpeptide having an ability to activate HLA-A2 restricted Cytotoxic Tlymphocytes 12 Met Leu Pro Leu Glu Cys Gln Tyr Leu 1 5 13 9 PRTArtificial Designed peptide having an ability to activate HLA-A2restricted Cytotoxic T lymphocytes 13 Thr Leu Leu Arg Lys Glu Arg GlyVal 1 5 14 9 PRT Artificial Designed peptide having an ability toactivate HLA-A2 restricted Cytotoxic T lymphocytes 14 Arg Ile Ile TyrAsp Arg Lys Phe Leu 1 5 15 9 PRT Artificial Designed peptide having anability to activate HLA-A2 restricted Cytotoxic T lymphocytes 15 Ile IleTyr Asp Arg Lys Phe Leu Met 1 5 16 9 PRT Artificial Designed peptidehaving an ability to activate HLA-A2 restricted Cytotoxic T lymphocytes16 Gln Ile Leu Lys Gly Leu Leu Phe Leu 1 5 17 9 PRT Artificial Designedpeptide having an ability to activate HLA-A2 restricted Cytotoxic Tlymphocytes 17 Gly Leu Leu Phe Leu His Thr Arg Thr 1 5 18 9 PRTArtificial Designed peptide having an ability to activate HLA-A2restricted Cytotoxic T lymphocytes 18 Asp Leu Leu Ser His Ala Phe PheAla 1 5 19 9 PRT Artificial Designed peptide having an ability toactivate HLA-A2 restricted Cytotoxic T lymphocytes 19 Gln Gln Leu CysVal Tyr Trp Thr Val 1 5 20 9 PRT Artificial Designed peptide having anability to activate HLA-A2 restricted Cytotoxic T lymphocytes 20 Ser LeuLeu Tyr Leu Asn Gln Ser Val 1 5 21 9 PRT Artificial Designed peptidehaving an ability to activate HLA-A2 restricted Cytotoxic T lymphocytes21 Lys Val His Pro Val Ile Trp Ser Leu 1 5 22 9 PRT Artificial Designedpeptide having an ability to activate HLA-A2 restricted Cytotoxic Tlymphocytes 22 Asp Met Trp Thr Val Ile Ala Pro Val 1 5 23 9 PRTArtificial Designed peptide having an ability to activate HLA-A2restricted Cytotoxic T lymphocytes 23 Gln Leu Gly Gln Gly Asn Leu LysVal 1 5 24 9 PRT Artificial Designed peptide having an ability toactivate HLA-A2 restricted Cytotoxic T lymphocytes 24 Leu Leu Leu LysTyr Thr Glu Lys Leu 1 5 25 9 PRT Artificial Designed peptide having anability to activate HLA-A2 restricted Cytotoxic T lymphocytes 25 Thr MetLeu Pro His His Ala Gly Leu 1 5 26 9 PRT Artificial Designed peptidehaving an ability to activate HLA-A2 restricted Cytotoxic T lymphocytes26 Leu Leu Arg Arg Ala Asp Phe His Val 1 5 27 9 PRT Artificial Designedpeptide having an ability to activate HLA-A2 restricted Cytotoxic Tlymphocytes 27 Glu Leu Leu Asn Arg Tyr Leu Tyr Leu 1 5 28 9 PRTArtificial Designed peptide having an ability to activate HLA-A2restricted Cytotoxic T lymphocytes 28 Leu Leu Met Leu Gln Asn Ala LeuThr 1 5 29 9 PRT Artificial Designed peptide having an ability toactivate HLA-A2 restricted Cytotoxic T lymphocytes 29 Tyr Gln Leu ProAsp Trp Arg Leu Val 1 5 30 10 PRT Artificial Designed peptide having anability to activate HLA-A2 restricted Cytotoxic T lymphocytes 30 Leu MetLeu Gln Asn Ala Leu Thr Thr Met 1 5 10 31 10 PRT Artificial Designedpeptide having an ability to activate HLA-A2 restricted Cytotoxic Tlymphocytes 31 Leu Leu Met Leu Gln Asn Ala Leu Thr Thr 1 5 10 32 10 PRTArtificial Designed peptide having an ability to activate HLA-A2restricted Cytotoxic T lymphocytes 32 Thr Gln Leu Ala Thr Tyr Ser PheGlu Val 1 5 10 33 10 PRT Artificial Designed peptide having an abilityto activate HLA-A2 restricted Cytotoxic T lymphocytes 33 Leu Glu Trp TyrAsp Asp Phe Pro His Val 1 5 10 34 10 PRT Artificial Designed peptidehaving an ability to activate HLA-A2 restricted Cytotoxic T lymphocytes34 Phe Ser Gly Asp Val Met Ile His Pro Val 1 5 10 35 9 PRT ArtificialDesigned peptide having an ability to activate HLA-A2 restrictedCytotoxic T lymphocytes 35 Trp Ala Gln Glu Asp Pro Asn Ala Val 1 5 36 10PRT Artificial Designed peptide having an ability to activate HLA-A2restricted Cytotoxic T lymphocytes 36 Ala Val Leu Gly Arg His Lys MetHis Val 1 5 10 37 10 PRT Artificial Designed peptide having an abilityto activate HLA-A2 restricted Cytotoxic T lymphocytes 37 Arg Leu Trp GluVal Ala Thr Ala Arg Cys 1 5 10 38 9 PRT Artificial Designed peptidehaving an ability to activate HLA-A2 restricted Cytotoxic T lymphocytes38 Val Ala Trp Asn Pro Ser Pro Ala Val 1 5 39 9 PRT Artificial Designedpeptide having an ability to activate HLA-A2 restricted Cytotoxic Tlymphocytes 39 Asp Leu Leu Gln Asn Pro Leu Leu Val 1 5 40 10 PRTArtificial Designed peptide having an ability to activate HLA-A2restricted Cytotoxic T lymphocytes 40 Leu Leu Gln Asn Pro Leu Leu ValPro Val 1 5 10 41 10 PRT Artificial Designed peptide having an abilityto activate HLA-A2 restricted Cytotoxic T lymphocytes 41 Val Ile Phe HisPro Thr Gln Pro Trp Val 1 5 10 42 10 PRT Artificial Designed peptidehaving an ability to activate HLA-A2 restricted Cytotoxic T lymphocytes42 Asn Leu Val Arg Asp Asp Gly Ser Ala Val 1 5 10 43 9 PRT ArtificialDesigned peptide having an ability to activate HLA-A2 restrictedCytotoxic T lymphocytes 43 Arg Leu Phe Ala Phe Val Arg Phe Thr 1 5 44 10PRT Artificial Designed peptide having an ability to activate HLA-A2restricted Cytotoxic T lymphocytes 44 Val Val Gln Asn Phe Ala Lys GluPhe Val 1 5 10 45 270 PRT Homo sapiens 45 Met Ala Gly Ala Glu Asp TrpPro Gly Gln Gln Leu Glu Leu Asp Glu 1 5 10 15 Asp Glu Ala Ser Cys CysArg Trp Gly Ala Gln His Ala Gly Ala Arg 20 25 30 Glu Leu Ala Ala Leu TyrSer Pro Gly Lys Arg Leu Gln Glu Trp Cys 35 40 45 Ser Val Ile Leu Cys PheSer Leu Ile Ala His Asn Leu Val His Leu 50 55 60 Leu Leu Leu Ala Arg TrpGlu Asp Thr Pro Leu Val Ile Leu Gly Val 65 70 75 80 Val Ala Gly Ala LeuIle Ala Asp Phe Leu Ser Gly Leu Val His Trp 85 90 95 Gly Ala Asp Thr TrpGly Ser Val Glu Leu Pro Ile Val Gly Lys Ala 100 105 110 Phe Ile Arg ProPhe Arg Glu His His Ile Asp Pro Thr Ala Ile Thr 115 120 125 Arg His AspPhe Ile Glu Thr Asn Gly Asp Asn Cys Leu Val Thr Leu 130 135 140 Leu ProLeu Leu Asn Met Ala Tyr Lys Phe Arg Thr His Ser Pro Glu 145 150 155 160Ala Leu Glu Gln Leu Tyr Pro Trp Glu Cys Phe Val Phe Cys Leu Ile 165 170175 Ile Phe Gly Thr Phe Thr Asn Gln Ile His Lys Trp Ser His Thr Tyr 180185 190 Phe Gly Leu Pro Arg Trp Val Thr Leu Leu Gln Asp Trp His Val Ile195 200 205 Leu Pro Arg Lys His His Arg Ile His His Val Ser Pro His GluThr 210 215 220 Tyr Phe Cys Ile Thr Thr Gly Trp Leu Asn Tyr Pro Leu GluLys Ile 225 230 235 240 Gly Phe Trp Arg Arg Leu Glu Asp Leu Ile Gln GlyLeu Thr Gly Glu 245 250 255 Lys Pro Arg Ala Asp Asp Met Lys Trp Ala GlnLys Ile Lys 260 265 270 46 589 PRT Homo sapiens 46 Met Ser Arg Arg LeuLeu Pro Arg Ala Glu Lys Arg Arg Arg Arg Leu 1 5 10 15 Glu Gln Arg GlnGln Pro Asp Glu Gln Arg Arg Arg Ser Gly Ala Met 20 25 30 Val Lys Met AlaAla Ala Gly Gly Gly Gly Gly Gly Gly Arg Tyr Tyr 35 40 45 Gly Gly Gly SerGlu Gly Gly Arg Ala Pro Lys Arg Leu Lys Thr Asp 50 55 60 Asn Ala Gly AspGln His Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala 65 70 75 80 Gly Ala AlaGly Gly Gly Gly Gly Gly Glu Asn Tyr Asp Asp Pro His 85 90 95 Lys Thr ProAla Ser Pro Val Val His Ile Arg Gly Leu Ile Asp Gly 100 105 110 Val ValGlu Ala Asp Leu Val Glu Ala Leu Gln Glu Phe Gly Pro Ile 115 120 125 SerTyr Val Val Val Met Pro Lys Lys Arg Gln Ala Leu Val Glu Phe 130 135 140Glu Asp Val Leu Gly Ala Cys Asn Ala Val Asn Tyr Ala Ala Asp Asn 145 150155 160 Gln Ile Tyr Ile Ala Gly His Pro Ala Phe Val Asn Tyr Ser Thr Ser165 170 175 Gln Lys Ile Ser Arg Pro Gly Asp Ser Asp Asp Ser Arg Ser ValAsn 180 185 190 Ser Val Leu Leu Phe Thr Ile Leu Asn Pro Ile Tyr Ser IleThr Thr 195 200 205 Asp Val Leu Tyr Thr Ile Cys Asn Pro Cys Gly Pro ValGln Arg Ile 210 215 220 Val Ile Phe Arg Lys Asn Gly Val Gln Ala Met ValGlu Phe Asp Ser 225 230 235 240 Val Gln Ser Ala Gln Arg Ala Lys Ala SerLeu Asn Gly Ala Asp Ile 245 250 255 Tyr Ser Gly Cys Cys Thr Leu Lys IleGlu Tyr Ala Lys Pro Thr Arg 260 265 270 Leu Asn Val Phe Lys Asn Asp GlnAsp Thr Trp Asp Tyr Thr Asn Pro 275 280 285 Asn Leu Ser Gly Gln Gly AspPro Gly Ser Asn Pro Asn Lys Arg Gln 290 295 300 Arg Gln Pro Pro Leu LeuGly Asp His Pro Ala Glu Tyr Gly Gly Pro 305 310 315 320 His Gly Gly TyrHis Ser His Tyr His Asp Glu Gly Tyr Gly Pro Pro 325 330 335 Pro Pro HisTyr Glu Gly Arg Arg Met Gly Pro Pro Val Gly Gly His 340 345 350 Arg ArgGly Pro Ser Arg Tyr Gly Pro Gln Tyr Gly His Pro Pro Pro 355 360 365 ProPro Pro Pro Pro Glu Tyr Gly Pro His Ala Asp Ser Pro Val Leu 370 375 380Met Val Tyr Gly Leu Asp Gln Ser Lys Met Asn Gly Asp Arg Val Phe 385 390395 400 Asn Val Phe Cys Leu Tyr Gly Asn Val Glu Lys Val Lys Phe Met Lys405 410 415 Ser Lys Pro Gly Ala Ala Met Val Glu Met Ala Asp Gly Tyr AlaVal 420 425 430 Asp Arg Ala Ile Thr His Leu Asn Asn Asn Phe Met Phe GlyGln Lys 435 440 445 Leu Asn Val Cys Val Ser Lys Gln Pro Ala Ile Met ProGly Gln Ser 450 455 460 Tyr Gly Leu Glu Asp Gly Ser Cys Ser Tyr Lys AspPhe Ser Glu Ser 465 470 475 480 Arg Asn Asn Arg Phe Ser Thr Pro Glu GlnAla Ala Lys Asn Arg Ile 485 490 495 Gln His Pro Ser Asn Val Leu His PhePhe Asn Ala Pro Leu Glu Val 500 505 510 Thr Glu Glu Asn Phe Phe Glu IleCys Asp Glu Leu Gly Val Lys Arg 515 520 525 Pro Ser Ser Val Lys Val PheSer Gly Lys Ser Glu Arg Ser Ser Ser 530 535 540 Gly Leu Leu Glu Trp GluSer Lys Ser Asp Ala Leu Glu Thr Leu Gly 545 550 555 560 Phe Leu Asn HisTyr Gln Met Lys Asn Pro Asn Gly Pro Tyr Pro Tyr 565 570 575 Thr Leu LysLeu Cys Phe Ser Thr Ala Gln His Ala Ser 580 585 47 549 PRT Homo sapiens47 Met Ala Ser Met Arg Glu Ser Asp Thr Gly Leu Trp Leu His Asn Lys 1 510 15 Leu Gly Ala Thr Asp Glu Leu Trp Ala Pro Pro Ser Ile Ala Ser Leu 2025 30 Leu Thr Ala Ala Ile Ile Asp Asn Ile Arg Leu Cys Phe His Gly Leu 3540 45 Ser Ser Ala Ser Leu Leu Thr Ala Ala Val Ile Asp Asn Ile Arg Leu 5055 60 Cys Phe His Gly Leu Ser Ser Ala Val Lys Leu Lys Leu Leu Leu Gly 6570 75 80 Thr Leu His Leu Pro Arg Arg Thr Val Asp Glu Met Lys Gly Ala Leu85 90 95 Met Glu Ile Ile Gln Leu Ala Ser Leu Asp Ser Asp Pro Trp Val Leu100 105 110 Met Val Ala Asp Ile Leu Lys Ser Phe Pro Asp Thr Gly Ser LeuAsn 115 120 125 Leu Glu Leu Glu Glu Gln Asn Pro Asn Val Gln Asp Ile LeuGly Glu 130 135 140 Leu Arg Glu Lys Val Gly Glu Cys Glu Ala Ser Ala MetLeu Pro Leu 145 150 155 160 Glu Cys Gln Tyr Leu Asn Lys Asn Ala Leu ThrThr Leu Ala Gly Pro 165 170 175 Leu Thr Pro Pro Val Lys His Phe Gln LeuLys Arg Lys Pro Lys Ser 180 185 190 Ala Thr Leu Arg Ala Glu Leu Leu GlnLys Ser Thr Glu Thr Ala Gln 195 200 205 Gln Leu Lys Arg Ser Ala Gly ValPro Phe His Ala Lys Gly Arg Gly 210 215 220 Leu Leu Arg Lys Met Asp ThrThr Thr Pro Leu Lys Gly Ile Pro Lys 225 230 235 240 Gln Ala Pro Phe ArgSer Pro Thr Ala Pro Ser Val Phe Ser Pro Thr 245 250 255 Gly Asn Arg ThrPro Ile Pro Pro Ser Arg Thr Leu Leu Arg Lys Glu 260 265 270 Arg Gly ValLys Leu Leu Asp Ile Ser Glu Leu Asp Met Val Gly Ala 275 280 285 Gly ArgGlu Ala Lys Arg Arg Arg Lys Thr Leu Asp Ala Glu Val Val 290 295 300 GluLys Pro Ala Lys Glu Glu Thr Val Val Glu Asn Ala Thr Pro Asp 305 310 315320 Tyr Ala Ala Gly Leu Val Ser Thr Gln Lys Leu Gly Ser Leu Asn Asn 325330 335 Glu Pro Ala Leu Pro Ser Thr Ser Tyr Leu Pro Ser Thr Pro Ser Val340 345 350 Val Pro Ala Ser Ser Tyr Ile Pro Ser Ser Glu Thr Pro Pro AlaPro 355 360 365 Ser Ser Arg Glu Ala Ser Arg Pro Pro Glu Glu Pro Ser AlaPro Ser 370 375 380 Pro Thr Leu Pro Ala Gln Phe Lys Gln Arg Ala Pro MetTyr Asn Ser 385 390 395 400 Gly Leu Ser Pro Ala Thr Pro Thr Pro Ala AlaPro Thr Ser Pro Leu 405 410 415 Thr Pro Thr Thr Pro Pro Ala Val Ala ProThr Thr Gln Thr Pro Pro 420 425 430 Val Ala Met Val Ala Pro Gln Thr GlnAla Pro Ala Gln Gln Gln Pro 435 440 445 Lys Lys Asn Leu Ser Leu Thr ArgGlu Gln Met Phe Ala Ala Gln Glu 450 455 460 Met Phe Lys Thr Ala Asn LysVal Thr Arg Pro Glu Lys Ala Leu Ile 465 470 475 480 Leu Gly Phe Met AlaGly Ser Arg Glu Asn Pro Cys Gln Glu Gln Gly 485 490 495 Asp Val Ile GlnIle Lys Leu Ser Glu His Thr Glu Asp Leu Pro Lys 500 505 510 Ala Asp GlyGln Gly Ser Thr Thr Met Leu Val Asp Thr Val Phe Glu 515 520 525 Met AsnTyr Ala Thr Gly Gln Trp Thr Arg Phe Lys Lys Tyr Lys Pro 530 535 540 MetThr Asn Val Ser 545 48 118 PRT Homo sapiens 48 Met Ser Gly Gly Ser SerCys Ser Gln Thr Pro Ser Arg Ala Ile Pro 1 5 10 15 Ala Thr Arg Arg ValVal Leu Gly Asp Gly Val Gln Leu Pro Pro Gly 20 25 30 Asp Tyr Ser Thr ThrPro Gly Gly Thr Leu Phe Ser Thr Thr Pro Gly 35 40 45 Gly Thr Arg Ile IleTyr Asp Arg Lys Phe Leu Met Glu Cys Arg Asn 50 55 60 Ser Pro Val Thr LysThr Pro Pro Arg Asp Leu Pro Thr Ile Pro Gly 65 70 75 80 Val Thr Ser ProSer Ser Asp Glu Pro Pro Met Glu Ala Ser Gln Ser 85 90 95 His Leu Arg AsnSer Pro Glu Asp Lys Arg Ala Gly Gly Glu Glu Ser 100 105 110 Gln Phe GluMet Asp Ile 115 49 779 PRT Homo sapiens 49 Met Glu Pro Gly Arg Gly AlaGly Pro Ala Gly Met Ala Glu Pro Arg 1 5 10 15 Ala Lys Ala Ala Arg ProGly Pro Gln Arg Phe Leu Arg Arg Ser Val 20 25 30 Val Glu Ser Asp Gln GluGlu Pro Pro Gly Leu Glu Ala Ala Glu Ala 35 40 45 Pro Gly Pro Gln Pro ProGln Pro Leu Gln Arg Arg Val Leu Leu Leu 50 55 60 Cys Lys Thr Arg Arg LeuIle Ala Glu Arg Ala Arg Gly Arg Pro Ala 65 70 75 80 Ala Pro Ala Pro AlaAla Leu Val Ala Gln Pro Gly Ala Pro Gly Ala 85 90 95 Pro Ala Asp Ala GlyPro Glu Pro Val Gly Thr Gln Glu Pro Gly Pro 100 105 110 Asp Pro Ile AlaAla Ala Val Glu Thr Ala Pro Ala Pro Asp Gly Gly 115 120 125 Pro Arg GluGlu Ala Ala Ala Thr Val Arg Lys Glu Asp Glu Gly Ala 130 135 140 Ala GluAla Lys Pro Glu Pro Gly Arg Thr Arg Arg Asp Glu Pro Glu 145 150 155 160Glu Glu Glu Asp Asp Glu Asp Asp Leu Lys Ala Val Ala Thr Ser Leu 165 170175 Asp Gly Arg Phe Leu Lys Phe Asp Ile Glu Leu Gly Arg Gly Ser Phe 180185 190 Lys Thr Val Tyr Lys Gly Leu Asp Thr Glu Thr Trp Val Glu Val Ala195 200 205 Trp Cys Glu Leu Gln Asp Arg Lys Leu Thr Lys Leu Glu Arg GlnArg 210 215 220 Phe Lys Glu Glu Ala Glu Met Leu Lys Gly Leu Gln His ProAsn Ile 225 230 235 240 Val Arg Phe Tyr Asp Phe Trp Glu Ser Ser Ala LysGly Lys Arg Cys 245 250 255 Ile Val Leu Val Thr Glu Leu Met Thr Ser GlyThr Leu Lys Thr Tyr 260 265 270 Leu Lys Arg Phe Lys Val Met Lys Pro LysVal Leu Arg Ser Trp Cys 275 280 285 Arg Gln Ile Leu Lys Gly Leu Leu PheLeu His Thr Arg Thr Pro Pro 290 295 300 Ile Ile His Arg Asp Leu Lys CysAsp Asn Ile Phe Ile Thr Gly Pro 305 310 315 320 Thr Gly Ser Val Lys IleGly Asp Leu Gly Leu Ala Thr Leu Lys Arg 325 330 335 Ala Ser Phe Ala LysSer Val Ile Gly Thr Pro Glu Phe Met Ala Pro 340 345 350 Glu Met Tyr GluGlu His Tyr Asp Glu Ser Val Asp Val Tyr Ala Phe 355 360 365 Gly Met CysMet Leu Glu Met Ala Thr Ser Glu Tyr Pro Tyr Ser Glu 370 375 380 Cys GlnAsn Ala Ala Gln Ile Tyr Arg Lys Val Thr Cys Gly Ile Lys 385 390 395 400Pro Ala Ser Phe Glu Lys Val His Asp Pro Glu Ile Lys Glu Ile Ile 405 410415 Gly Glu Cys Ile Cys Lys Asn Lys Glu Glu Arg Tyr Glu Ile Lys Asp 420425 430 Leu Leu Ser His Ala Phe Phe Ala Glu Asp Thr Gly Val Arg Val Glu435 440 445 Leu Ala Glu Glu Asp His Gly Arg Lys Ser Thr Ile Ala Leu ArgLeu 450 455 460 Trp Val Glu Asp Pro Lys Lys Leu Lys Gly Lys Pro Lys AspAsn Gly 465 470 475 480 Ala Ile Glu Phe Thr Phe Asp Leu Glu Lys Glu ThrPro Asp Glu Val 485 490 495 Ala Gln Glu Met Ile Glu Ser Gly Phe Phe HisGlu Ser Asp Val Lys 500 505 510 Ile Val Ala Lys Ser Ile Arg Asp Arg ValAla Leu Ile Gln Trp Arg 515 520 525 Arg Glu Arg Ile Trp Pro Ala Leu GlnPro Lys Glu Gln Gln Asp Val 530 535 540 Gly Ser Pro Asp Lys Ala Arg GlyPro Pro Val Pro Leu Gln Val Gln 545 550 555 560 Val Thr Tyr His Ala GlnAla Gly Gln Pro Gly Pro Pro Glu Pro Glu 565 570 575 Glu Pro Glu Ala AspGln His Leu Leu Pro Pro Thr Leu Pro Thr Ser 580 585 590 Ala Thr Ser LeuAla Ser Asp Ser Thr Phe Asp Ser Gly Gln Gly Ser 595 600 605 Thr Val TyrSer Asp Ser Gln Ser Ser Gln Gln Ser Val Met Leu Gly 610 615 620 Ser LeuAla Asp Ala Ala Pro Ser Pro Ala Gln Cys Val Cys Ser Pro 625 630 635 640Pro Val Ser Glu Gly Pro Val Leu Pro Gln Ser Leu Pro Ser Leu Gly 645 650655 Ala Tyr Gln Gln Pro Thr Ala Ala Pro Pro Pro Leu Ala Gln Pro Thr 660665 670 Pro Leu Pro Gln Val Leu Ala Pro Gln Pro Val Val Pro Leu Gln Pro675 680 685 Val Pro Pro His Leu Pro Pro Tyr Leu Ala Pro Ala Ser Gln ValGly 690 695 700 Ala Pro Ala Gln Leu Lys Pro Leu Gln Met Pro Gln Ala ProLeu Gln 705 710 715 720 Pro Leu Ala Gln Val Pro Pro Gln Met Pro Pro IlePro Val Val Pro 725 730 735 Pro Ile Thr Pro Leu Ala Gly Ile Asp Gly LeuPro Pro Ala Leu Pro 740 745 750 Asp Leu Pro Thr Ala Thr Val Pro Pro ValPro Pro Pro Gln Tyr Phe 755 760 765 Ser Pro Ala Val Ile Leu Pro Ser ArgThr Arg 770 775 50 1087 PRT Homo sapiens 50 Met Asp Leu Tyr Ser Thr ProAla Ala Ala Leu Asp Arg Phe Val Ala 1 5 10 15 Arg Lys Leu Gln Pro ArgLys Glu Phe Val Glu Lys Ala Arg Arg Ala 20 25 30 Leu Gly Ala Leu Ala AlaAla Leu Arg Glu Arg Gly Gly Arg Leu Gly 35 40 45 Ala Ala Ala Pro Arg ValLeu Lys Thr Val Lys Gly Gly Ser Ser Gly 50 55 60 Arg Gly Thr Ala Leu LysGly Gly Cys Asp Ser Glu Leu Val Ile Phe 65 70 75 80 Leu Asp Cys Phe LysSer Tyr Val Asp Gln Arg Ala Arg Arg Ala Glu 85 90 95 Ile Leu Ser Glu MetArg Ala Ser Leu Glu Ser Trp Trp Gln Asn Pro 100 105 110 Val Pro Gly LeuArg Leu Thr Phe Pro Glu Gln Ser Val Pro Gly Ala 115 120 125 Leu Gln PheArg Leu Thr Ser Val Asp Leu Glu Asp Trp Met Asp Val 130 135 140 Ser LeuVal Pro Ala Phe Asn Val Leu Gly Gln Ala Gly Ser Gly Val 145 150 155 160Lys Pro Lys Pro Gln Val Tyr Ser Thr Leu Leu Asn Ser Gly Cys Gln 165 170175 Gly Gly Glu His Ala Ala Cys Phe Thr Glu Leu Arg Arg Asn Phe Val 180185 190 Asn Ile Arg Pro Ala Lys Leu Lys Asn Leu Ile Leu Leu Val Lys His195 200 205 Trp Tyr His Gln Val Cys Leu Gln Gly Leu Trp Lys Glu Thr LeuPro 210 215 220 Pro Val Tyr Ala Leu Glu Leu Leu Thr Ile Phe Ala Trp GluGln Gly 225 230 235 240 Cys Lys Lys Asp Ala Phe Ser Leu Ala Glu Gly LeuArg Thr Val Leu 245 250 255 Gly Leu Ile Gln Gln His Gln His Leu Cys ValPhe Trp Thr Val Asn 260 265 270 Tyr Gly Phe Glu Asp Pro Ala Val Gly GlnPhe Leu Gln Arg Gln Leu 275 280 285 Lys Arg Pro Arg Pro Val Ile Leu AspPro Ala Asp Pro Thr Trp Asp 290 295 300 Leu Gly Asn Gly Ala Ala Trp HisTrp Asp Leu Leu Ala Gln Glu Ala 305 310 315 320 Ala Ser Cys Tyr Asp HisPro Cys Phe Leu Arg Gly Met Gly Asp Pro 325 330 335 Val Gln Ser Trp LysGly Pro Gly Leu Pro Arg Ala Gly Cys Ser Gly 340 345 350 Leu Gly His ProIle Gln Leu Asp Pro Asn Gln Lys Thr Pro Glu Asn 355 360 365 Ser Lys SerLeu Asn Ala Val Tyr Pro Arg Ala Gly Ser Lys Pro Pro 370 375 380 Ser CysPro Ala Pro Gly Pro Thr Gly Ala Ala Ser Ile Val Pro Ser 385 390 395 400Val Pro Gly Met Ala Leu Asp Leu Ser Gln Ile Pro Thr Lys Glu Leu 405 410415 Asp Arg Phe Ile Gln Asp His Leu Lys Pro Ser Pro Gln Phe Gln Glu 420425 430 Gln Val Lys Lys Ala Ile Asp Ile Ile Leu Arg Cys Leu His Glu Asn435 440 445 Cys Val His Lys Ala Ser Arg Val Ser Lys Gly Gly Ser Phe GlyArg 450 455 460 Gly Thr Asp Leu Arg Asp Gly Cys Asp Val Glu Leu Ile IlePhe Leu 465 470 475 480 Asn Cys Phe Thr Asp Tyr Lys Asp Gln Gly Pro ArgArg Ala Glu Ile 485 490 495 Leu Asp Glu Met Arg Ala His Val Glu Ser TrpTrp Gln Asp Gln Val 500 505 510 Pro Ser Leu Ser Leu Gln Phe Pro Glu GlnAsn Val Pro Glu Ala Leu 515 520 525 Gln Phe Gln Leu Val Ser Thr Ala LeuLys Ser Trp Thr Asp Val Ser 530 535 540 Leu Leu Pro Ala Phe Asp Ala ValGly Gln Leu Ser Ser Gly Thr Lys 545 550 555 560 Pro Asn Pro Gln Val TyrSer Arg Leu Leu Thr Ser Gly Cys Gln Glu 565 570 575 Gly Glu His Lys AlaCys Phe Ala Glu Leu Arg Arg Asn Phe Met Asn 580 585 590 Ile Arg Pro ValLys Leu Lys Asn Leu Ile Leu Leu Val Lys His Trp 595 600 605 Tyr Arg GlnVal Ala Ala Gln Asn Lys Gly Lys Gly Pro Ala Pro Ala 610 615 620 Ser LeuPro Pro Ala Tyr Ala Leu Glu Leu Leu Thr Ile Phe Ala Trp 625 630 635 640Glu Gln Gly Cys Arg Gln Asp Cys Phe Asn Met Ala Gln Gly Phe Arg 645 650655 Thr Val Leu Gly Leu Val Gln Gln His Gln Gln Leu Cys Val Tyr Trp 660665 670 Thr Val Asn Tyr Ser Thr Glu Asp Pro Ala Met Arg Met His Leu Leu675 680 685 Gly Gln Leu Arg Lys Pro Arg Pro Leu Val Leu Asp Pro Ala AspPro 690 695 700 Thr Trp Asn Val Gly His Gly Ser Trp Glu Leu Leu Ala GlnGlu Ala 705 710 715 720 Ala Ala Leu Gly Met Gln Ala Cys Phe Leu Ser ArgAsp Gly Thr Ser 725 730 735 Val Gln Pro Trp Asp Val Met Pro Ala Leu LeuTyr Gln Thr Pro Ala 740 745 750 Gly Asp Leu Asp Lys Phe Ile Ser Glu PheLeu Gln Pro Asn Arg Gln 755 760 765 Phe Leu Ala Gln Val Asn Lys Ala ValAsp Thr Ile Cys Ser Phe Leu 770 775 780 Lys Glu Asn Cys Phe Arg Asn SerPro Ile Lys Val Ile Lys Val Val 785 790 795 800 Lys Gly Gly Ser Ser AlaLys Gly Thr Ala Leu Arg Gly Arg Ser Asp 805 810 815 Ala Asp Leu Val ValPhe Leu Ser Cys Phe Ser Gln Phe Thr Glu Gln 820 825 830 Gly Asn Lys ArgAla Glu Ile Ile Ser Glu Ile Arg Ala Gln Leu Glu 835 840 845 Ala Cys GlnGln Glu Arg Gln Phe Glu Val Lys Phe Glu Val Ser Lys 850 855 860 Trp GluAsn Pro Arg Val Leu Ser Phe Ser Leu Thr Ser Gln Thr Met 865 870 875 880Leu Asp Gln Ser Val Asp Phe Asp Val Leu Pro Ala Phe Asp Ala Leu 885 890895 Gly Gln Leu Val Ser Gly Ser Arg Pro Ser Ser Gln Val Tyr Val Asp 900905 910 Leu Ile His Ser Tyr Ser Asn Ala Gly Glu Tyr Ser Thr Cys Phe Thr915 920 925 Glu Leu Gln Arg Asp Phe Ile Ile Ser Arg Pro Thr Lys Leu LysSer 930 935 940 Leu Ile Arg Leu Val Lys His Trp Tyr Gln Gln Cys Thr LysIle Ser 945 950 955 960 Lys Gly Arg Gly Ser Leu Pro Pro Gln His Gly LeuGlu Leu Leu Thr 965 970 975 Val Tyr Ala Trp Glu Gln Gly Arg Lys Asp SerGln Phe Asn Met Ala 980 985 990 Glu Gly Phe Arg Thr Val Leu Glu Leu ValThr Gln Tyr Arg Gln Leu 995 1000 1005 Cys Ile Tyr Trp Thr Ile Asn TyrAsn Ala Lys Asp Lys Thr Val 1010 1015 1020 Gly Asp Phe Leu Lys Gln GlnLeu Gln Lys Pro Arg Pro Ile Ile 1025 1030 1035 Leu Asp Pro Ala Asp ProThr Gly Asn Leu Gly His Asn Ala Arg 1040 1045 1050 Trp Asp Leu Leu AlaLys Glu Ala Ala Ala Cys Thr Ser Ala Leu 1055 1060 1065 Cys Cys Met GlyArg Asn Gly Ile Pro Ile Gln Pro Trp Pro Val 1070 1075 1080 Lys Ala AlaVal 1085 51 216 PRT Homo sapiens 51 Met Lys Ser Ile Ser Leu Leu Arg TyrGln Glu Glu Ser Lys Thr Leu 1 5 10 15 Ser Leu Val Ser Arg Asp Ala LysPro Leu Glu Val Tyr Ser Val Asp 20 25 30 Phe Met Val Asp Asn Ala Gln LeuGly Phe Leu Val Ser Asp Arg Asp 35 40 45 Arg Asn Leu Met Val Tyr Met TyrLeu Pro Glu Ala Lys Glu Ser Phe 50 55 60 Gly Gly Met Arg Leu Leu Arg ArgAla Asp Phe His Val Gly Ala His 65 70 75 80 Val Asn Thr Phe Trp Arg ThrPro Cys Arg Gly Ala Thr Glu Gly Leu 85 90 95 Ser Lys Lys Ser Val Val TrpGlu Asn Lys His Ile Thr Trp Phe Ala 100 105 110 Thr Leu Asp Gly Gly IleGly Leu Leu Leu Pro Met Gln Glu Lys Thr 115 120 125 Tyr Arg Arg Leu LeuMet Leu Gln Asn Ala Leu Thr Thr Met Leu Pro 130 135 140 His His Ala GlyLeu Asn Pro Arg Ala Phe Arg Met Leu His Val Asp 145 150 155 160 Arg ArgThr Leu Gln Asn Ala Val Arg Asn Val Leu Asp Gly Glu Leu 165 170 175 LeuAsn Arg Tyr Leu Tyr Leu Ser Thr Met Glu Arg Ser Glu Leu Ala 180 185 190Lys Lys Ile Gly Thr Thr Pro Asp Ile Ile Leu Asp Asp Leu Leu Glu 195 200205 Thr Asp Arg Val Thr Ala His Phe 210 215 52 634 PRT Homo sapiens 52Met Ala Ser Ala Arg Ile Gly Asp Glu Tyr Ala Glu Asp Ser Ser Asp 1 5 1015 Glu Glu Asp Ile Arg Asn Thr Val Gly Asn Val Pro Leu Glu Trp Tyr 20 2530 Asp Asp Phe Pro His Val Gly Tyr Asp Leu Asp Gly Arg Arg Ile Tyr 35 4045 Lys Pro Leu Arg Thr Arg Asp Glu Leu Asp Gln Phe Leu Asp Lys Met 50 5560 Asp Asp Pro Asp Tyr Trp Arg Thr Val Gln Asp Pro Met Thr Gly Arg 65 7075 80 Asp Leu Arg Leu Thr Asp Glu Gln Val Ala Leu Val Arg Arg Leu Gln 8590 95 Ser Gly Gln Phe Gly Asp Val Gly Phe Asn Pro Tyr Glu Pro Ala Val100 105 110 Asp Phe Phe Ser Gly Asp Val Met Ile His Pro Val Thr Asn ArgPro 115 120 125 Ala Asp Lys Arg Ser Phe Ile Pro Ser Leu Val Glu Lys GluLys Val 130 135 140 Ser Arg Met Val His Ala Ile Lys Met Gly Trp Ile GlnPro Arg Arg 145 150 155 160 Pro Arg Asp Pro Thr Pro Ser Phe Tyr Asp LeuTrp Ala Gln Glu Asp 165 170 175 Pro Asn Ala Val Leu Gly Arg His Lys MetHis Val Pro Ala Pro Lys 180 185 190 Leu Ala Leu Pro Gly His Ala Glu SerTyr Asn Pro Pro Pro Glu Tyr 195 200 205 Leu Leu Ser Glu Glu Glu Arg LeuAla Trp Glu Gln Gln Glu Pro Gly 210 215 220 Glu Arg Lys Leu Ser Phe LeuPro Arg Lys Phe Pro Ser Leu Arg Ala 225 230 235 240 Val Pro Ala Tyr GlyArg Phe Ile Gln Glu Arg Phe Glu Arg Cys Leu 245 250 255 Asp Leu Tyr LeuCys Pro Arg Gln Arg Lys Met Arg Val Asn Val Asp 260 265 270 Pro Glu AspLeu Ile Pro Lys Leu Pro Arg Pro Arg Asp Leu Gln Pro 275 280 285 Phe ProThr Cys Gln Ala Leu Val Tyr Arg Gly His Ser Asp Leu Val 290 295 300 ArgCys Leu Ser Val Ser Pro Gly Gly Gln Trp Leu Val Ser Gly Ser 305 310 315320 Asp Asp Gly Ser Leu Arg Leu Trp Glu Val Ala Thr Ala Arg Cys Val 325330 335 Arg Thr Val Pro Val Gly Gly Val Val Lys Ser Val Ala Trp Asn Pro340 345 350 Ser Pro Ala Val Cys Leu Val Ala Ala Ala Val Glu Asp Ser ValLeu 355 360 365 Leu Leu Asn Pro Ala Leu Gly Asp Arg Leu Val Ala Gly SerThr Asp 370 375 380 Gln Leu Leu Ser Ala Phe Val Pro Pro Glu Glu Pro ProLeu Gln Pro 385 390 395 400 Ala Arg Trp Leu Glu Ala Ser Glu Glu Glu ArgGln Val Gly Leu Arg 405 410 415 Leu Arg Ile Cys His Gly Lys Pro Val ThrGln Val Thr Trp His Gly 420 425 430 Arg Gly Asp Tyr Leu Ala Val Val LeuAla Thr Gln Gly His Thr Gln 435 440 445 Val Leu Ile His Gln Leu Ser ArgArg Arg Ser Gln Ser Pro Phe Arg 450 455 460 Arg Ser His Gly Gln Val GlnArg Val Ala Phe His Pro Ala Arg Pro 465 470 475 480 Phe Leu Leu Val AlaSer Gln Arg Ser Val Arg Leu Tyr His Leu Leu 485 490 495 Arg Gln Glu LeuThr Lys Lys Leu Met Pro Asn Cys Lys Trp Val Ser 500 505 510 Ser Leu AlaVal His Pro Ala Gly Asp Asn Val Ile Cys Gly Ser Tyr 515 520 525 Asp SerLys Leu Val Trp Phe Asp Leu Asp Leu Ser Thr Lys Pro Tyr 530 535 540 ArgMet Leu Arg His His Lys Lys Ala Leu Arg Ala Val Ala Phe His 545 550 555560 Pro Arg Tyr Pro Leu Phe Ala Ser Gly Ser Asp Asp Gly Ser Val Ile 565570 575 Val Cys His Gly Met Val Tyr Asn Asp Leu Leu Gln Asn Pro Leu Leu580 585 590 Val Pro Val Lys Val Leu Lys Gly His Val Leu Thr Arg Asp LeuGly 595 600 605 Val Leu Asp Val Ile Phe His Pro Thr Gln Pro Trp Val PheSer Ser 610 615 620 Gly Ala Asp Gly Thr Val Arg Leu Phe Thr 625 630 53142 PRT Homo sapiens 53 Met Ala Thr Lys Ile Asp Lys Glu Ala Cys Arg AlaAla Tyr Asn Leu 1 5 10 15 Val Arg Asp Asp Gly Ser Ala Val Ile Trp ValThr Phe Lys Tyr Asp 20 25 30 Gly Ser Thr Ile Val Pro Gly Glu Gln Gly AlaGlu Tyr Gln His Phe 35 40 45 Ile Gln Gln Cys Thr Asp Asp Val Arg Leu PheAla Phe Val Arg Phe 50 55 60 Thr Thr Gly Asp Ala Met Ser Lys Arg Ser LysPhe Ala Leu Ile Thr 65 70 75 80 Trp Ile Gly Glu Asn Val Ser Gly Leu GlnArg Ala Lys Thr Gly Thr 85 90 95 Asp Lys Thr Leu Val Lys Glu Val Val GlnAsn Phe Ala Lys Glu Phe 100 105 110 Val Ile Ser Asp Arg Lys Glu Leu GluGlu Asp Phe Ile Lys Ser Glu 115 120 125 Leu Lys Lys Ala Gly Gly Ala AsnTyr Asp Ala Gln Thr Glu 130 135 140 54 1775 DNA Homo sapiens polyA_site(1775)..(1775) 54 atctcgccgc ggttccgcgg ccctgccgcc gccgccnttt ngcagagcgcaccgggccga 60 tcgggcgagt ggccatggcg ggcgccgagg actggccggg ccagcagctggagctggacg 120 aggacgaggc gtcttgttgc cgctggggcg cgcagcacgc cggggcccgcgagctggctg 180 cgctctactc gccaggcaag cgcctccagg agtggtgctc tgtgatcctgtgcttcagcc 240 tcatcgccca caacctggtc catctcctgc tgctggcccg ctgggaggacacacccctcg 300 tcatactcgg tgttgttgca ggggctctca ttgctgactt cttgtctggcctggtacact 360 ggggtgctga cacatggggc tctgtggagc tgcccattgt ggggaaggctttcatccgac 420 ccttccggga gcaccacatt gacccgacag ctatcacacg gcacgacttcatcgagacca 480 acggggacaa ctgcctggtg acactgctgc cgctgctaaa catggcctacaagttccgca 540 cccacagccc tgaagccctg gagcagctat acccctggga gtgcttcgtcttctgcctga 600 tcatcttcgg caccttcacc aaccagatcc acaagtggtc gcacacgtactttgggctgc 660 cacgctgggt caccctcctg caggactggc atgtcatcct gccacgtaaacaccatcgca 720 tccaccacgt ctcaccccac gagacctact tctgcatcac cacaggctggctcaactacc 780 ctctggagaa gataggcttc tggcgacgcc tggaggacct catccagggcctgacgggcg 840 agaagcctcg ggcagatgac atgaaatggg cccagaagat caaataacttctccgagcct 900 gctacctggt tgccaacctt ccctagcccc caaaccgaag ccatctgccaaattccagcc 960 tctttgagct ggcccctcca gatggagagg acatctcctg ggctgggcccaggtacccca 1020 gcccacccct catgacacag aatacttgag ccactgattt ttcatttctttttttttttt 1080 tcctcggccc ctcctcagcc acctgagttg ctctatctgc aagcctgactctgccagcct 1140 cccctggtag agaggaggtt tacccactcc ctgcacgcct gccgtccctgccccgctggg 1200 caagcccttc agtgtggctg gcgttggggc cagtgagttg cctctttccctccttgtctg 1260 gccccagtgg tctggggagc ccccaggcac acctaagcgt cgtggagcattgttctgcca 1320 cagccctgca tactgacccc gggaggctgg gcaggtggac agccccagccaccaccttca 1380 gcctagcctg tcccccaagg atggtgaagc tcagcagggg tctgagggtagccggccaga 1440 agaggctgga acctcctgct caagtctaga cccctacttc tctgctgcccccaccctgcc 1500 agagctgatg tttccaatac caagatgtct tcacagggca cagcccctgcagagcatctt 1560 ggtcatttgg aagaggacac ggtatcccct ctggccagag tatgtcagagaaggaagagt 1620 agggcttttt tgttttgttt ttttttaaag gtgcttgctt gtttaatgtaaataatagaa 1680 agccttaata tcttttctgt aacacggagt aatattttaa tgtcatgttttggatgtaca 1740 taatatattt ataacaaagc agcaagagtc tactt 1775 55 2097 DNAHomo sapiens polyA_site (2097)..(2097) 55 gccgccatgt cgcggaggctgctgccccgg gcggagaagc ggcgtcggcg gctggagcag 60 aggcagcagc cggacgagcagcggaggcgg tcgggagcga tggtgaagat ggcggcggcg 120 ggcggcggag gcggcggtggccgctactac ggcggcggca gtgagggcgg ccgggcccct 180 aagcggctca agactgacaacgccggcgac cagcacggag gcggcggcgg tggcggtgga 240 ggagccgggg cggcgggcggcggcggcggt ggggagaact acgatgaccc gcacaaaacc 300 cctgcctccc cagttgtccacatcaggggc ctgattgacg gtgtggtgga agcagacctt 360 gtggaggcct tgcaggagtttggacccatc agctatgtgg tggtaatgcc taaaaagaga 420 caagcactgg tggagtttgaagatgtgttg ggggcttgca acgcagtgaa ctacgcagcc 480 gacaaccaaa tatacattgctggtcaccca gcttttgtca actactctac cagccagaag 540 atctcccgcc ctggggactcggatgactcc cggagcgtga acagtgtgct tctctttacc 600 atcctgaacc ccatttattcgatcaccacg gatgttcttt acactatctg taatccttgt 660 ggccctgtcc agagaattgtcattttcagg aagaatggag ttcaggcgat ggtggaattt 720 gactcagttc aaagtgcccagcgggccaag gcctctctca atggggctga tatctattct 780 ggctgttgca ctctgaagatcgaatacgca aagcctacac gcttgaatgt gttcaagaat 840 gatcaggata cttgggactacacaaacccc aatctcagtg gacaaggtga ccctggcagc 900 aaccccaaca aacgccagaggcagccccct ctcctgggag atcaccccgc agaatatgga 960 gggccccacg gtgggtaccacagccattac catgatgagg gctacgggcc ccccccacct 1020 cactacgaag ggagaaggatgggtccacca gtggggggtc accgtcgggg cccaagtcgc 1080 tacggccccc agtatgggcaccccccaccc cctcccccac cacccgagta tggccctcac 1140 gccgacagcc ctgtgctcatggtctatggc ttggatcaat ctaagatgaa cggtgaccga 1200 gtcttcaatg tcttctgcttatatggcaat gtggagaagg tgaaattcat gaaaagcaag 1260 ccgggggccg ccatggtggagatggctgat ggctacgctg tagaccgggc cattacccac 1320 ctcaacaaca acttcatgtttgggcagaag ctgaatgtct gtgtctccaa gcagccagcc 1380 atcatgcctg gtcagtcatacgggttggaa gacgggtctt gcagttacaa agacttcagt 1440 gaatcccgga acaatcggttctccacccca gagcaggcag ccaagaaccg catccagcac 1500 cccagcaacg tgctgcacttcttcaacgcc ccgctggagg tgaccgagga gaacttcttt 1560 gagatctgcg atgagctgggagtgaagcgg ccatcttctg tgaaagtatt ctcaggcaaa 1620 agtgagcgca gctcctctggactgctggag tgggaatcca agagcgatgc cctggagact 1680 ctgggcttcc tgaaccattaccagatgaaa aacccaaatg gtccataccc ttacactctg 1740 aagttgtgtt tctccactgctcagcacgcc tcctaattag gtgcctagga agagtcccat 1800 ctgagcagga agacatttctctttccttta tgccattttt tgtttttgtt atttgcaaaa 1860 gatcttgtat tccttttttttttttttttt ttttttaaat gctaggtttg tagaggctta 1920 cttaacctta atggaaacgctggaaatctg cagggggagg gaaaggggaa ctgttatctc 1980 ccaagattaa ccttcacttttaaaaaatta ttggacatgt gaattttttt tttcctggtc 2040 atacatttgg gctgcccatgtactcttggc ccatttcaat aaaattgttt ggaaaat 2097 56 2243 DNA Homo sapienspolyA_site (2243)..(2243) 56 atggcgtcca tgcgggagag cgacacgggc ctgtggctgcacaacaagct gggggccacg 60 gacgagctgt angcgccgcc cagcatcgcg tccctgctcacggccgcaat catcgacaac 120 atccgtctct gcttccatgg cctctcgtcg gcgtccctgctcacggccgc ggtcatcgac 180 aacatccgtc tctgcttcca tggcctctcg tcggcagtgaagctcaagtt gctactcggg 240 acgctgcacc tcccgcgccg cacggtggac gagatgaagggcgccctaat ggagatcatc 300 cagctcgcca gcctcgactc ggacccctgg gtgctcatggtcgccgacat cttgaagtcc 360 tttccggaca caggctcgct taacctggag ctggaggagcagaatcccaa cgttcaggat 420 attttgggag aacttagaga aaaggtgggt gagtgtgaagcgtctgccat gctgccactg 480 gagtgccagt acttgaacaa aaacgccctg acgaccctcgcgggacccct cactcccccg 540 gtgaagcatt ttcagttaaa gcggaaaccc aagagcgccacgctgcgggc ggagctgctg 600 cagaagtcca cggagaccgc ccagcagttg aagcggagcgccggggtgcc cttccacgcc 660 aagggccggg ggctgctgcg gaagatggac accaccaccccactcaaagg catcccgaag 720 caggcgccct tcagaagccc cacggcgccc agcgtcttcagccccacagg gaaccggacc 780 cccatcccgc cttccaggac gctgctgcgg aaggaacgaggtgtgaagct gctggacatc 840 tctgagctgg atatggttgg cgctggccga gaggcgaagcggagaaggaa gactctcgat 900 gcggaggtgg tggagaagcc ggccaaggag gaaacggtggtggagaacgc caccccggac 960 tacgcagccg gcctggtgtc cacgcagaaa cttgggtccctgaacaatga gcctgcgctg 1020 ccctccacga gctaccttcc ctccacgccc agcgtggttcccgcctcctc ctacatcccc 1080 agctccgaga cgcccccagc cccatcttcc cgggaagccagccgcccacc agaggagccc 1140 agcgccccga gccccacgtt gccagcgcag ttcaagcagcgggcgcccat gtacaacagc 1200 ggcctgagcc ctgccacacc cacgcctgcg gcgcccacctcgcctctgac acccaccaca 1260 cctccggctg tcgcccctac cactcagaca cccccggttgccatggtggc cccgcagacc 1320 caggcccctg ctcagcagca gcctaagaag aacctgtccctcacgagaga gcagatgttc 1380 gctgcccagg agatgttcaa gacggccaac aaagtcacgcggcccgagaa ggccctcatc 1440 ctgggcttca tggccggctc ccgagagaac ccgtgccaggagcaggggga cgtgatccag 1500 atcaagctga gcgagcacac ggaggacctg cccaaggcggacggccaggg tagcacaacc 1560 atgctggtgg acacagtgtt tgagatgaac tatgccacgggccagtggac gcgcttcaag 1620 aagtacaagc ccatgaccaa tgtgtcctag aaccacctgcctcacagctg gccgtcactt 1680 gtgggggtcc acgggacgat ggctttgcca gcttaaagtaaccggatggc ggacacctgg 1740 cccccgaggt cccccggccg ccgccctgct gctgacccagcctgttttaa gttctggatg 1800 catttctctg gggtatttgg ggcttatttt taaaattttaatatgggttc ttttttgtgt 1860 gatttaagac actttttgga ctcaacgtta catttttgaatgtagtaagt aaattaacca 1920 aaaaagttac aacttcctaa ttttagtgac agctctgcctgttagactct tactttttaa 1980 aatcttttct attttccctc gctggggcag tgccctcctacccccagggt tgaggggacc 2040 aaggtggcac ggtggtactg ggggtgcggc agggacacccgaccacacca gagcgtggga 2100 gacggtgggc cttgtcccct gcctgtgcct gcctgggagttttgtattca tcttttgtat 2160 agttgtggac atttaagaca gtctttgggt acctattttcattgtaaaac tatctgaacc 2220 attaaagtcg agcttttcta aag 2243 57 831 DNAHomo sapiens polyA_site (831)..(831) 57 gcgggagggc agcgagaggt tcgcgggtgcagcgcacagg agaccatgtc cgggggcagc 60 agctgcagcc agaccccaag ccgggccatccccgccactc gccgggtggt gctcggcgac 120 ggcgtgcagc tcccgcccgg ggactacagcacgacccccg gcggcacgct cttcagcacc 180 accccgggag gtaccaggat catctatgaccggaaattcc tgatggagtg tcggaactca 240 cctgtgacca aaacaccccc aagggatctgcccaccattc cgggggtcac cagcccttcc 300 agtgatgagc cccccatgga agccagccagagccacctgc gcaatagccc agaagataag 360 cgggcgggcg gtgaagagtc acagtttgagatggacattt aaagcaccag ccatcgtgtg 420 gagcactacc aaggggcccc tcagggccttcctgggagga gtcccaccag ccaggcctta 480 tgaaagtgat catactgggc aggcgttggcgtggggtcgg acaccccagc cctttctccc 540 tcactcaggg cacctgcccc ctcctcttcgtgaacaccag cagatacctc cttgtgcctc 600 cactgatgca ggagctgcca ccccaaggggagtgacccct gccagcacac cctgcagcca 660 agggccagga agtggacaag aacgaacccttccttccgaa tgatcagcag ttccagcccc 720 tcgctgctgg gggcgcaacc accccttccttaggttgatg tgcttgggaa agctccctcc 780 ccctccttcc ccaagagagg aaataaaagccaccttcgcc ctagggccaa g 831 58 2404 DNA Homo sapiens 58 cacgcgtaagcttgggcccc tcgagggatc ctctagagcg gccgccgaga cgtccccggc 60 acgctgatggagcccgggcg cggcgcgggg cccgcgggca tggcggagcc tcgggcgaag 120 gcggcgcggccggggcccca gcgctttctg cggcgcagcg tggtagagtc ggaccaggag 180 gagccgccgggcttggaggc agccgaggcg ccgggcccgc agcccccgca gcccctgcag 240 cgccgggtgcttctgctctg caagacgcgc cgcctcatcg cggagcgcgc ccgcggacgc 300 cccgccgcccccgcgcccgc agcgctggta gcgcagccgg gagcccccgg agcccccgcg 360 gacgccggccccgagcccgt gggcacgcag gagcccggcc cggaccccat cgcagccgct 420 gtcgaaaccgcgcctgcccc cgacggcggc cccagggagg aggcggcggc taccgtgagg 480 aaggaggatgagggggcggc cgaggcgaag cctgagcccg ggcgcactcg ccgggacgag 540 cccgaagaggaggaggacga cgaggacgac ctcaaggccg tggccacctc tctggacggc 600 cgcttcctcaagttcgacat cgagctgggc cgcggttcct tcaagacggt ctacaagggg 660 ctggacacggagacctgggt ggaggtggcc tggtgtgagc tgcaggaccg gaagctcacc 720 aagctggagcggcagcggtt caaggaagag gctgagatgc tgaaaggcct gcagcacccc 780 aacatcgtgcgcttctacga cttctgggag tccagcgcca agggcaagcg gtgcattgtg 840 ctggtgacggagcttatgac ctcagggacg ctgaagacat acctgaagcg gttcaaggtg 900 atgaagcccaaggttctccg cagctggtgc cggcagatcc tgaagggcct gctgttcctg 960 cacacaaggacgccacccat catccaccga gacctgaaat gtgacaatat tttcatcacc 1020 ggaccaactgggtctgtgaa gattggcgac ttgggcctgg ccactctgaa aagagcgtca 1080 tttgccaaaagtgtgatagg tactcccgag ttcatggcgc ccgagatgta cgaggagcac 1140 tacgatgagtccgtggacgt ctatgccttt gggatgtgca tgctggagat ggccacctcg 1200 gagtacccctactcggagtg ccagaatgcg gcccagatct accgcaaggt cacctgtggt 1260 atcaagccggccagctttga gaaagtgcac gatcctgaaa tcaaggagat tattggggag 1320 tgtatctgcaaaaacaagga ggaaaggtac gagatcaaag acctgctgag ccacgccttc 1380 ttcgcagaggacacaggcgt gagggtggag ctcgcggagg aggaccacgg caggaagtcc 1440 accatcgccctgaggctctg ggtggaagac cccaagaaac tgaagggaaa gcccaaggac 1500 aatggagccatagagttcac cttcgacctg gagaaggaga cgccggatga agtggcccaa 1560 gagatgattgagtctggatt cttccacgag agtgacgtca agatcgtggc caagtccatc 1620 cgtgaccgcgtggccttgat ccagtggcgg cgggagagga tctggcccgc gctgcagccc 1680 aaggagcagcaggatgtggg cagcccggac aaggccaggg gtccgccggt gcccctgcag 1740 gtccaggtgacctaccatgc acaggctggg cagcccgggc caccagagcc cgaggagccg 1800 gaggccgaccagcacctcct gccacctacg ttgccgacca gcgccacctc cctggcctcg 1860 gacagcaccttcgacagcgg ccagggctct accgtgtact cagactcgca gagcagccag 1920 cagagcgtgatgcttggctc ccttgccgac gcagcgccgt ccccggccca gtgtgtgtgc 1980 agcccccctgtgagcgaggg gcccgtcctg ccgcagagcc tgccctcgct gggggcctac 2040 cagcagcccacggctgcacc tcctccgctg gcccagccga cacccctgcc gcaggtcctg 2100 gccccacagcccgtggtccc cctccagccg gttccccccc acctgccacc gtacctggct 2160 ccagcctcccaggtgggggc ccccgctcag ctgaagcccc tccagatgcc acaggcgccc 2220 ctgcagccgcttgctcaagt ccctccgcag atgcccccga ttcctgttgt gccccccatc 2280 acgcccctggcgggaatcga cggcctccct ccggccctcc cagacctgcc gaccgcgact 2340 gtgcctcccgtgccaccacc tcagtatttc tctccagccg tgatcttgcc gagccggacg 2400 cgtg 2404 596707 DNA Homo sapiens polyA_site (6707)..(6707) 59 cccaagtttg gggaagacaggaactgcagc gcccctcccc gtttcacgcc acgcgcggga 60 ccgaggacct aggacctggccagctgggcg tggttcggag agccgggcgg gaaaacgaaa 120 ccagaaatcc gaaggccgcgccagagccct gcttcccctt gcatctgcgc cgggaggcca 180 tggacttgta cagcaccccggccgctgcgc tggacaggtt cgtggccaga aagctgcagc 240 cgcggaagga gttcgtagagaaggcgcggc gcgctctggg cgccctggcc gctgctctga 300 gggagcgcgg gggccgcctcggtgctgctg ccccgcgggt gctgaaaact gtcaagggag 360 gctcctcggg ccggggcacagctctcaagg gtggctgtga ttctgaactt gtcatcttcc 420 tcgactgctt caagagctatgtggaccaga gggcccgccg tgcagagatc ctcagtgaga 480 tgcgggcatc gctggaatcctggtggcaga acccagtccc tggtctgaga ctcacgtttc 540 ctgagcagag cgtgcctggggccctgcagt tccgcctgac atccgtagat cttgaggact 600 ggatggatgt tagcctggtgcctgccttca atgtcctggg tcaggccggc tccggcgtca 660 aacccaagcc acaagtctactctaccctcc tcaacagtgg ctgccaaggg ggcgagcatg 720 cggcctgctt cacagagctgcggaggaact ttgtgaacat tcgcccagcc aagttgaaga 780 acctaatctt gctggtgaagcactggtacc accaggtgtg cctacagggg ttgtggaagg 840 agacgctgcc cccggtctatgccctggaat tgctgaccat cttcgcctgg gagcagggct 900 gtaagaagga tgctttcagcctagccgaag gcctccgaac tgtcctgggc ctgatccaac 960 agcatcagca cctgtgtgttttctggactg tcaactatgg cttcgaggac cctgcagttg 1020 ggcagttctt gcagcggcagcttaagagac ccaggcctgt gatcctggac ccagctgacc 1080 ccacatggga cctggggaatggggcagcct ggcactggga tttgctagcc caggaggcag 1140 catcctgcta tgaccacccatgctttctga gggggatggg ggacccagtg cagtcttgga 1200 aggggccggg ccttccacgtgctggatgct caggtttggg ccaccccatc cagctagacc 1260 ctaaccagaa gacccctgaaaacagcaaga gcctcaatgc tgtgtaccca agagcaggga 1320 gcaaacctcc ctcatgcccagctcctggcc ccactggggc agccagcatc gtcccctctg 1380 tgccgggaat ggccttggacctgtctcaga tccccaccaa ggagctggac cgcttcatcc 1440 aggaccacct gaagccgagcccccagttcc aggagcaggt gaaaaaggcc atcgacatca 1500 tcttgcgctg cctccatgagaactgtgttc acaaggcctc aagagtcagt aaagggggct 1560 catttggccg gggcacagacctaagggatg gctgtgatgt tgaactcatc atcttcctca 1620 actgcttcac ggactacaaggaccaggggc cccgccgcgc agagatcctt gatgagatgc 1680 gagcgcacgt agaatcctggtggcaggacc aggtgcccag cctgagcctt cagtttcctg 1740 agcagaatgt gcctgaggctctgcagttcc agctggtgtc cacagccctg aagagctgga 1800 cggatgttag cctgctgcctgccttcgatg ctgtggggca gctcagttct ggcaccaaac 1860 caaatcccca ggtctactcaaggctcctca ccagtggctg ccaggagggc gagcataagg 1920 cctgcttcgc agagctgcggaggaacttca tgaacattcg ccctgtcaag ctgaagaacc 1980 tgattctgct ggtgaagcactggtaccgcc aggttgcggc tcagaacaaa ggaaaaggac 2040 cagcccctgc ctctctgcccccagcctatg ccctggagct cctcaccatc tttgcctggg 2100 agcagggctg caggcaggattgtttcaaca tggcccaagg cttccggacg gtgctggggc 2160 tcgtgcaaca gcatcagcagctctgtgtct actggacggt caactatagc actgaggacc 2220 cagccatgag aatgcaccttcttggccagc ttcgaaaacc cagacccctg gtcctggacc 2280 ccgctgatcc cacctggaacgtgggccacg gtagctggga gctgttggcc caggaagcag 2340 cagcgctggg gatgcaggcctgctttctga gtagagacgg gacatctgtg cagccctggg 2400 atgtgatgcc agccctcctttaccaaaccc cagctgggga ccttgacaag ttcatcagtg 2460 aatttctcca gcccaaccgccagttcctgg cccaggtgaa caaggccgtt gataccatct 2520 gttcattttt gaaggaaaactgcttccgga attctcccat caaagtgatc aaggtggtca 2580 agggtggctc ttcagccaaaggcacagctc tgcgaggccg ctcagatgcc gacctcgtgg 2640 tgttcctcag ctgcttcagccagttcactg agcagggcaa caagcgggcc gagatcatct 2700 ccgagatccg agcccagctggaggcatgtc aacaggagcg gcagttcgag gtcaagtttg 2760 aagtctccaa atgggagaatccccgcgtgc tgagcttctc actgacatcc cagacgatgc 2820 tggaccagag tgtggactttgatgtgctgc cagcctttga cgccctaggc cagctggtct 2880 ctggctccag gcccagctctcaagtctacg tcgacctcat ccacagctac agcaatgcgg 2940 gcgagtactc cacctgcttcacagagctac aacgggactt catcatctct cgccctacca 3000 agctgaagag cctgatccggctggtgaagc actggtacca gcagtgtacc aagatctcca 3060 aggggagagg ctccctacccccacagcacg ggctggaact cctgactgtg tatgcctggg 3120 agcagggccg gaaggactcccagttcaaca tggctgaggg cttccgcacg gtcctggagc 3180 tggtcaccca gtaccgccagctctgtatct actggaccat caactacaac gccaaggaca 3240 agactgttgg agacttcctgaaacagcagc ttcagaagcc caggcctatc atcctggatc 3300 cggctgaccc gacaggcaacctgggccaca atgcccgctg ggacctgctg gccaaggaag 3360 ctgcagcctg cacatctgccctgtgctgca tgggacggaa tggcatcccc atccagccat 3420 ggccagtgaa ggctgctgtgtgaagttgag aaaatcagcg gtcctactgg atgaagagaa 3480 gatggacacc agccctcagcatgaggaaat tcagggtccc ctaccagatg agagagattg 3540 tgtacatgtg tgtgtgagcacatgtgtgca tgtgtgtgca cacgtgtgca tgtgtgtgtt 3600 ttagtgaatc tgctctcccagctcacacac tcccctgcct cccatggctt acacactagg 3660 atccagactc catggtttgacaccagcctg cgtttgcagc ttctctgtca cttccatgac 3720 tctatcctca taccaccactgctgcttccc acccagctga gaatgccccc tcctccctga 3780 ctcctctctg cccatgcaaattagctcaca tctttcctcc tgctgcaatc catcccttcc 3840 tcccattggc ctctccttgccaaatctaaa tactttatat agggatggca gagagttccc 3900 atctcatctg tcagccacagtcatttggta ctggctacct ggagccttat cttctgaagg 3960 gttttaaaga atggccaattagctgagaag aattatctaa tcaattagtg atgtctgcca 4020 tggatgcagt agaggaaagtggtggtacaa gtgccatgat tgattagcaa tgtctgcact 4080 ggatatggaa aaaagaaggtgcttgcaggt ttacagtgta tatgtgggct attgaagagc 4140 cctctgagct cggttgctagcaggagagca tgcccatatt ggcttacttt gtctgccaca 4200 gacacagaca gagggagttgggacatgcat gctatgggga ccctcttgtt ggacacctaa 4260 ttggatgcct cttcatgagaggcctccttt tcttcacctt ttatgctgca ctcctcccct 4320 agtttacaca tcttgatgctgtggctcagt ttgccttcct gaatttttat tgggtccctg 4380 ttttctctcc taacatgctgagattctgca tccccacagc ctaaactgag ccagtggcca 4440 aacaaccgtg ctcagcctgtttctctctgc cctctagagc aaggcccacc aggtccatcc 4500 aggaggctct cctgacctcaagtccaacaa cagtgtccac actagtcaag gttcagccca 4560 gaaaacagaa agcactctaggaatcttagg cagaaaggga ttttatctaa atcactggaa 4620 aggctggagg agcagaaggcagaggccacc actggactat tggtttcaat attagaccac 4680 tgtagccgaa tcagaggccagagagcagcc actgctactg ctaatgccac cactacccct 4740 gccatcactg ccccacatggacaaaactgg agtcgagacc taggttagat tcctgcaacc 4800 acaaacatcc atcagggatggccagctgcc agagctgcgg gaagacggat cccacctccc 4860 tttcttagca gaatctaaattacagccaga cctctggctg cagaggagtc tgagacatgt 4920 atgattgaat gggtgccaagtgccaggggg cggagtcccc agcagatgca tcctggccat 4980 ctgttgcgtg gatgagggagtgggtctatc tcagaggaag gaacaggaaa caaagaaagg 5040 aagccactga acatcccttctctgctccac aggagtgtct tagacagcct gactctccac 5100 aaaccactgt taaaacttacctgctaggaa tgctagattg aatgggatgg gaagagcctt 5160 ccctcattat tgtcattcttggagagaggt gagcaaccaa gggaagctcc tctgattcac 5220 ctagaacctg ttctctgccgtctttggctc agcctacaga gactagagta ggtgaaggga 5280 cagaggacag ggcttctaatacctgtgcca tattgacagc ctccatccct gtcccccatc 5340 ttggtgctga accaacgctaagggcacctt cttagactca cctcatcgat actgcctggt 5400 aatccaaagc tagaactctcaggaccccaa actccacctc ttggattggc cctggctgct 5460 gccacacaca tatccaagagctcagggcca gttctggtgg gcagcagaga cctgctctgc 5520 caagttgtcc agcagcagagtggccctggc ctgggcatca caagccagtg atgctcctgg 5580 gaagaccagg tggcaggtcgcagttgggta ccttccattc ccaccacaca gactctgggc 5640 ctccccgcaa aatggctccagaattagagt aattatgaga tggtgggaac cagagcaact 5700 caggtgcatg atacaaggagaggttgtcat ctgggtaggg cagagaggag ggcttgctca 5760 tctgaacagg ggtgtatttcattccaggcc ctcagtcttt ggcaatggcc accctggtgt 5820 tggcatattg gccccactgtaacttttggg ggcttcccgg tctagccaca ccctcggatg 5880 gaaagacttg actgcataaagatgtcagtt ctccctgagt tgattgatag gcttaatggt 5940 caccctaaaa acacccacatatgcttttcg atggaaccag ataagttgac gctaaagttc 6000 ttatggaaaa atacacacgcaatagctagg aaaacacagg gaaagaagag ttctgagcag 6060 ggcctagtct tagccaatattaaaacatac tatgaagcct ctgatactta aacagcatgg 6120 cgctggtacg taaatagaccaatgcagtta ggtggctctt tccaagactc tggggaaaaa 6180 agtagtaaaa agctaaatgcaatcaatcag caattgaaag ctaagtgaga gagccagagg 6240 gcctccttgg tggtaaaagagggttgcatt tcttgcagcc agaaggcaga gaaagtgaag 6300 accaagtcca gaactgaatcctaagaaatg caggactgca aagaaattgg tgtgtgtgtg 6360 tgtgtgtgtg tgtttaatttttaaaaagtt tttattgaga tacaagtcaa taccataaag 6420 ctctcaccct tctaaagtgtacaattcagt ggtgtgagta tattcataag atttatactt 6480 ggtgtctatt cataagacttatatccagca tattcataac tagagccata tcacagatgc 6540 attcatcata ataattccagacattttcat caccctaaaa ggaaaccctg aaacccatta 6600 gcagtcattc cccattcctccaacccattc tctccctaat ccctagaaac caccaatctg 6660 ctgtgtattt catctattgccaacatttca tataaatggc atcatac 6707 60 769 DNA Homo sapiens polyA_site(769)..(769) 60 gcgtcaagaa cttcatcctg gcagccgacg tcatgaagag catttcgctgctgcgctacc 60 aggaggaaag caagacgctg agcctggtgt cgcgggatgc caagcccctggaggtgtaca 120 gcgtggactt catggtggac aatgcccagc tgggttttct ggtgtctgaccgcgaccgca 180 acctcatggt gtacatgtac ctgcccgaag ccaaggagag tttcgggggcatgcgcctgc 240 tgcgtcgggc agacttccac gtgggtgccc acgtgaacac gttctggaggaccccgtgcc 300 ggggggccac tgaagggctc agcaaaaagt cggtcgtgtg ggagaataagcacatcacgt 360 ggtttgccac cctggacggc ggcatcgggc tgctgctgcc catgcaggagaagacctacc 420 ggcggctgct gatgctgcag aacgcgctga ccaccatgct gccacaccacgccggcctca 480 acccccgcgc cttccggatg ctgcacgtgg accgccgcac cctccagaatgccgtgcgca 540 acgtgctgga tggggagctg ctcaaccgct acctgtacct gagcaccatggagcgcagcg 600 agctagccaa gaagatcggc accacaccag acataatcct ggacgacttgctggagacgg 660 accgcgtcac cgcccacttc tagccccgtg gatgccgtca ccaccagcacacggaactac 720 ctcccacccc ctttttgtac aaaacacaag gaaaaacatt ttttgcttg 76961 2039 DNA Homo sapiens polyA_site (2039)..(2039) 61 gagggccacagtgggattaa aaagaccact gaggagcagg tgcaggccag cactccttgc 60 ccgaggacagagatggcgag cgcccggatt ggggatgagt atgcggagga cagctctgat 120 gaggaggacatccggaacac ggtgggcaac gtgcccttgg agtggtacga tgacttcccc 180 cacgtgggctacgacctgga tggcaggcgc atctacaagc ccctgcggac ccgggatgag 240 ctggaccagttcctggacaa gatggacgat cctgactact ggcgcaccgt gcaggacccg 300 atgacagggcgggacctgag actgacggat gagcaggtgg ccctggtgcg gcggctgcag 360 agtggccagtttggggatgt gggcttcaac ccctatgagc cggctgtcga cttcttcagc 420 ggggacgtcatgatccaccc ggtgaccaac cgcccggccg acaagcgcag cttcatcccc 480 tccctggtggagaaggagaa ggtctctcgc atggtgcacg ccatcaagat gggctggatc 540 cagcctcgccggccccgaga ccccaccccc agcttctatg acctgtgggc ccaggaggac 600 cccaacgccgtgctcgggcg ccacaagatg cacgtacctg ctcccaagct ggccctgcca 660 ggccacgccgagtcgtacaa cccaccccct gaatacctgc tcagcgagga ggagcgcttg 720 gcgtgggaacagcaggagcc aggcgagagg aagctgagct ttttgccacg caagttcccg 780 agcctgcgggccgtgcctgc ctacggacgc ttcatccagg aacgcttcga gcgctgcctt 840 gacctgtacctgtgcccacg gcagcgcaag atgagggtga atgtagaccc tgaggacctc 900 atccccaagctgcctcggcc gagggacctg cagcccttcc ccacgtgcca ggccctggtc 960 tacaggggccacagtgacct tgtccggtgc ctcagtgtct ctcctggggg ccagtggctg 1020 gtttcaggctctgacgacgg ctccctgcgg ctctgggagg tggccactgc ccgctgtgtg 1080 aggactgttcccgtgggggg cgtggtgaag agtgtggcct ggaaccccag ccccgctgtc 1140 tgcctggtggctgcagccgt ggaggactcg gtgctgctgc tgaacccagc tctgggggac 1200 cggctggtggcgggcagcac agatcagctg ttgagcgcct tcgtcccgcc tgaggagccc 1260 cccttgcagccggcccgctg gctggaggcc tcagaggagg agcgccaagt gggcctgcgg 1320 ctgcgcatctgccacgggaa gccagtgacg caggtgacct ggcacgggcg tggggactac 1380 ctggccgtggtgctggccac ccaaggccac acccaggtgc tgattcacca gctgagccgt 1440 cgccgcagccagagtccgtt ccgccgcagc cacggacagg tgcagcgagt ggccttccac 1500 cctgcccggcccttcctgtt ggtggcgtcc cagcgcagcg tccgcctcta ccacctgctg 1560 cgccaggagctcaccaagaa gctgatgccc aactgcaagt gggtgtccag cctggcggtg 1620 caccctgcaggtgacaacgt catctgtggg agctacgata gcaagctggt gtggtttgac 1680 ctggatctttccaccaagcc atacaggatg ctgagacacc acaagaaggc tctgcgggct 1740 gtggccttccacccgcggta cccactcttt gcgtcaggct cggacgacgg cagtgtcatc 1800 gtctgccatggcatggtgta caatgacctt ctgcagaacc ccttgctggt gcccgtcaag 1860 gtgctgaagggacacgtgct gacccgagat ctgggagtgc tggacgtcat cttccacccc 1920 acccagccgtgggtcttctc ctcgggggca gacgggactg tccgcctctt cacctagctg 1980 ttctgcctgcctggggctgg ggtggtcgtg ctgaagtcaa cagagccttt accctgtgc 2039 62 1822 DNAHomo sapiens polyA_site (1822)..(1822) 62 cggcctgcag ctcgcaggcgccgcgtagcc gtcgccaccg ccgccagccc gtgcgccctc 60 ggcggtaccc gccgcgctcccatccccgcc gccggccagg ggcgcgctcg gccgccccgg 120 acagtgtccc gctgcggctccgcggcgatg gccaccaaga tcgacaaaga ggcttgccgg 180 gcggcgtaca acctggtgcgcgacgacggc tcggccgtca tctgggtgac ttttaaatat 240 gacggctcca ccatcgtccccggcgagcag ggagcggagt accagcactt catccagcag 300 tgcacagatg acgtccggttgtttgccttc gtgcgcttca ccaccgggga tgccatgagc 360 aagaggtcca agtttgccctcatcacgtgg atcggtgaga acgtcagcgg gctgcagcgc 420 gccaaaaccg ggacggacaagaccctggtg aaggaggtcg tacagaattt cgctaaggag 480 tttgtgatca gtgatcggaaggagctggag gaagatttca tcaagagcga gctgaagaag 540 gcggggggag ccaattacgacgcccagacg gagtaacccc agcccccgcc acaccacccc 600 ttgccaaagt catctgcctgctccccgggg gagaggaccg ccggcctcag ctactagccc 660 accagcccac cagggagaagagaagccatg agaggcagcg cccgccaccc tgtgtccaca 720 gcccccacct tcccgcttcccttagaaccc tgccgtgtcc tatctcatga cgctcatgga 780 acctctttct ttgatcttctttttcttttc tccccctctt ttttgttcta aagaaaagtc 840 attttgatgc aaggtcctgcctgccatcag atccgaggtg cctcctgcag tgaccccttt 900 tcctggcatt tctcttccacgcgacgaggt ctgcctagtg agatctgcat gacctcacgt 960 tgctttccag agcccgggcctattttgcca tctcagtttt cctgggccct gcttcctgtg 1020 taccactgag gggcagctgggccaggagct gtgcccggtg cctgcagcct tcataagcac 1080 acacgtccat tccctactaaggcccagacc tcctggtatc tgccccgggc tccctcatcc 1140 cacctccatc cggagttgcccaagatgcat gtccagcata ggcaggattg ctcggtggtg 1200 agaaggttag gtccggctcagactgaataa gaagagataa aatttgcctt aaaacttacc 1260 tggcagtggc tttgctgcacggtctgaaac cacctgttcc caccctcttg accgaaattt 1320 ccttgtgaca cagagaagggcaaaggtctt gagcccagag ttgacggagg gagtatttca 1380 gggttcactt caggggctcccaaagcgaca agatcgttag ggagagaggc ccagggtggg 1440 gactgggaat ttaaggagagctgggaacgg atcccttagg ttcaggaagc ttctgtgcaa 1500 gctgcgagga tggcttgggccgaagggttg ctctgcccgc cgcgctagct gtgagctgag 1560 caaagccctg ggctcacagcaccccaaaag cctgtggctt cagtcctgcg tctgcaccac 1620 acaatcaaaa ggatcgttttgttttgtttt taaagaaagg tgagattggc ttggttcttc 1680 atgagcacat ttgatatagctctttttctg tttttccttg ctcatttcgt tttggggaag 1740 aaatctgtac tgtattgggattgtaaagaa catctctgca ctcagacagt ttacagaaat 1800 aaatgttttt tttgtttttcag 1822 63 9 PRT Artificial Designed peptide based on the amino acidsequence of SEQ ID NO45 63 Ala Leu Tyr Ser Pro Gly Lys Arg Leu 1 5 64 10PRT Artificial Designed peptide based on the amino acid sequence of SEQID NO45 64 Ser Leu Ile Ala His Asn Leu Val His Leu 1 5 10 65 9 PRTArtificial Designed peptide based on the amino acid sequence of SEQ IDNO45 65 Leu Leu Leu Ala Arg Trp Glu Asp Thr 1 5 66 10 PRT ArtificialDesigned peptide based on the amino acid sequence of SEQ ID NO45 66 LeuLeu Ala Arg Trp Glu Asp Thr Pro Leu 1 5 10 67 10 PRT Artificial Designedpeptide based on the amino acid sequence of SEQ ID NO45 67 Ile Leu GlyVal Val Ala Gly Ala Leu Ile 1 5 10 68 10 PRT Artificial Designed peptidebased on the amino acid sequence of SEQ ID NO45 68 Ala Leu Ile Ala AspPhe Leu Ser Gly Leu 1 5 10 69 10 PRT Artificial Designed peptide basedon the amino acid sequence of SEQ ID NO45 69 Phe Leu Ser Gly Leu Val HisTrp Gly Ala 1 5 10 70 9 PRT Artificial Designed peptide based on theamino acid sequence of SEQ ID NO45 70 Cys Leu Val Thr Leu Leu Pro LeuLeu 1 5 71 9 PRT Artificial Designed peptide based on the amino acidsequence of SEQ ID NO45 71 Thr Leu Leu Pro Leu Leu Asn Met Ala 1 5 72 10PRT Artificial Designed peptide based on the amino acid sequence of SEQID NO45 72 Leu Leu Asn Met Ala Tyr Lys Phe Arg Thr 1 5 10 73 9 PRTArtificial Designed peptide based on the amino acid sequence of SEQ IDNO45 73 Gln Leu Tyr Pro Trp Glu Cys Phe Val 1 5 74 9 PRT ArtificialDesigned peptide based on the amino acid sequence of SEQ ID NO45 74 TyrPro Trp Glu Cys Phe Val Phe Cys 1 5 75 10 PRT Artificial Designedpeptide based on the amino acid sequence of SEQ ID NO45 75 Phe Val PheCys Leu Ile Ile Phe Gly Thr 1 5 10 76 10 PRT Artificial Designed peptidebased on the amino acid sequence of SEQ ID NO45 76 Phe Cys Leu Ile IlePhe Gly Thr Phe Thr 1 5 10 77 9 PRT Artificial Designed peptide based onthe amino acid sequence of SEQ ID NO45 77 Cys Leu Ile Ile Phe Gly ThrPhe Thr 1 5 78 10 PRT Artificial Designed peptide based on the aminoacid sequence of SEQ ID NO45 78 Ile Ile Phe Gly Thr Phe Thr Asn Gln Ile1 5 10 79 9 PRT Artificial Designed peptide based on the amino acidsequence of SEQ ID NO45 79 Gly Leu Pro Arg Trp Val Thr Leu Leu 1 5 80 10PRT Artificial Designed peptide based on the amino acid sequence of SEQID NO45 80 Trp Val Thr Leu Leu Gln Asp Trp His Val 1 5 10 81 9 PRTArtificial Designed peptide based on the amino acid sequence of SEQ IDNO45 81 Val Thr Leu Leu Gln Asp Trp His Val 1 5 82 9 PRT ArtificialDesigned peptide based on the amino acid sequence of SEQ ID NO45 82 ThrLeu Leu Gln Asp Trp His Val Ile 1 5 83 9 PRT Artificial Designed peptidebased on the amino acid sequence of SEQ ID NO45 83 Trp Leu Asn Tyr ProLeu Glu Lys Ile 1 5 84 9 PRT Artificial Designed peptide based on theamino acid sequence of SEQ ID NO45 84 Arg Leu Glu Asp Leu Ile Gln GlyLeu 1 5 85 9 PRT Artificial Designed peptide based on the amino acidsequence of SEQ ID NO46 85 Gly Leu Ile Asp Gly Val Val Glu Ala 1 5 86 9PRT Artificial Designed peptide based on the amino acid sequence of SEQID NO46 86 Gln Glu Phe Gly Pro Ile Ser Tyr Val 1 5 87 9 PRT ArtificialDesigned peptide based on the amino acid sequence of SEQ ID NO46 87 ValMet Pro Lys Lys Arg Gln Ala Leu 1 5 88 9 PRT Artificial Designed peptidebased on the amino acid sequence of SEQ ID NO46 88 Tyr Ile Ala Gly HisPro Ala Phe Val 1 5 89 10 PRT Artificial Designed peptide based on theamino acid sequence of SEQ ID NO46 89 Val Leu Leu Phe Thr Ile Leu AsnPro Ile 1 5 10 90 9 PRT Artificial Designed peptide based on the aminoacid sequence of SEQ ID NO46 90 Leu Leu Phe Thr Ile Leu Asn Pro Ile 1 591 9 PRT Artificial Designed peptide based on the amino acid sequence ofSEQ ID NO46 91 Val Leu Tyr Thr Ile Cys Asn Pro Cys 1 5 92 9 PRTArtificial Designed peptide based on the amino acid sequence of SEQ IDNO46 92 Ile Val Ile Phe Arg Lys Asn Gly Val 1 5 93 10 PRT ArtificialDesigned peptide based on the amino acid sequence of SEQ ID NO46 93 LysMet Asn Cys Asp Arg Val Phe Asn Val 1 5 10 94 9 PRT Artificial Designedpeptide based on the amino acid sequence of SEQ ID NO46 94 Met Asn CysAsp Arg Val Phe Asn Val 1 5 95 9 PRT Artificial Designed peptide basedon the amino acid sequence of SEQ ID NO46 95 Ile Met Pro Gly Gln Ser TyrGly Leu 1 5 96 9 PRT Artificial Designed peptide based on the amino acidsequence of SEQ ID NO46 96 Val Leu His Phe Phe Asn Ala Pro Leu 1 5 97 10PRT Artificial Designed peptide based on the amino acid sequence of SEQID NO46 97 Lys Leu Cys Phe Ser Thr Ala Gln His Ala 1 5 10 98 9 PRTArtificial Designed peptide based on the amino acid sequence of SEQ IDNO47 98 Ala Leu Met Glu Ile Ile Gln Leu Ala 1 5 99 9 PRT ArtificialDesigned peptide based on the amino acid sequence of SEQ ID NO47 99 SerLeu Asp Ser Asp Pro Trp Val Leu 1 5 100 10 PRT Artificial Designedpeptide based on the amino acid sequence of SEQ ID NO47 100 Leu Glu LeuGlu Glu Gln Asn Pro Asn Val 1 5 10 101 10 PRT Artificial Designedpeptide based on the amino acid sequence of SEQ ID NO47 101 Ala Met LeuPro Leu Glu Cys Gln Tyr Leu 1 5 10 102 9 PRT Artificial Designed peptidebased on the amino acid sequence of SEQ ID NO47 102 Tyr Leu Asn Lys AsnAla Leu Thr Thr 1 5 103 10 PRT Artificial Designed peptide based on theamino acid sequence of SEQ ID NO47 103 Thr Leu Ala Gly Pro Leu Thr ProPro Val 1 5 10 104 9 PRT Artificial Designed peptide based on the aminoacid sequence of SEQ ID NO47 104 Gln Gln Leu Lys Arg Ser Ala Gly Val 1 5105 9 PRT Artificial Designed peptide based on the amino acid sequenceof SEQ ID NO47 105 Gly Leu Leu Arg Lys Met Asp Thr Thr 1 5 106 10 PRTArtificial Designed peptide based on the amino acid sequence of SEQ IDNO47 106 Lys Leu Leu Asp Ile Ser Glu Leu Asp Met 1 5 10 107 10 PRTArtificial Designed peptide based on the amino acid sequence of SEQ IDNO47 107 Leu Leu Asp Ile Ser Glu Leu Asp Met Val 1 5 10 108 9 PRTArtificial Designed peptide based on the amino acid sequence of SEQ IDNO47 108 Tyr Leu Pro Ser Thr Pro Ser Val Val 1 5 109 9 PRT ArtificialDesigned peptide based on the amino acid sequence of SEQ ID NO47 109 ThrGln Thr Pro Pro Val Ala Met Val 1 5 110 9 PRT Artificial Designedpeptide based on the amino acid sequence of SEQ ID NO47 110 Ser Leu ThrArg Glu Gln Met Phe Ala 1 5 111 9 PRT Artificial Designed peptide basedon the amino acid sequence of SEQ ID NO47 111 Glu Met Phe Lys Thr AlaAsn Lys Val 1 5 112 9 PRT Artificial Designed peptide based on the aminoacid sequence of SEQ ID NO47 112 Lys Leu Ser Glu His Thr Glu Asp Leu 1 5113 10 PRT Artificial Designed peptide based on the amino acid sequenceof SEQ ID NO47 113 Thr Met Leu Val Asp Thr Val Phe Glu Met 1 5 10 114 9PRT Artificial Designed peptide based on the amino acid sequence of SEQID NO47 114 Met Leu Val Asp Thr Val Phe Glu Met 1 5 115 9 PRT ArtificialDesigned peptide based on the amino acid sequence of SEQ ID NO48 115 ValVal Leu Gly Asp Gly Val Gln Leu 1 5 116 10 PRT Artificial Designedpeptide based on the amino acid sequence of SEQ ID NO48 116 Gln Leu ProPro Gly Asp Tyr Ser Thr Thr 1 5 10 117 10 PRT Artificial Designedpeptide based on the amino acid sequence of SEQ ID NO48 117 Phe Leu MetGlu Cys Arg Asn Ser Pro Val 1 5 10 118 9 PRT Artificial Designed peptidebased on the amino acid sequence of SEQ ID NO49 118 Gly Met Ala Glu ProArg Ala Lys Ala 1 5 119 10 PRT Artificial Designed peptide based on theamino acid sequence of SEQ ID NO49 119 Val Leu Leu Leu Cys Lys Thr ArgArg Leu 1 5 10 120 9 PRT Artificial Designed peptide based on the aminoacid sequence of SEQ ID NO49 120 Leu Leu Leu Cys Lys Thr Arg Arg Leu 1 5121 9 PRT Artificial Designed peptide based on the amino acid sequenceof SEQ ID NO49 121 Lys Gly Leu Asp Thr Glu Thr Trp Val 1 5 122 10 PRTArtificial Designed peptide based on the amino acid sequence of SEQ IDNO49 122 Gly Leu Asp Thr Glu Thr Trp Val Glu Val 1 5 10 123 10 PRTArtificial Designed peptide based on the amino acid sequence of SEQ IDNO49 123 Glu Leu Gln Asp Arg Lys Leu Thr Lys Leu 1 5 10 124 9 PRTArtificial Designed peptide based on the amino acid sequence of SEQ IDNO49 124 Lys Thr Tyr Leu Lys Arg Phe Lys Val 1 5 125 10 PRT ArtificialDesigned peptide based on the amino acid sequence of SEQ ID NO49 125 ArgGln Ile Leu Lys Gly Leu Leu Phe Leu 1 5 10 126 9 PRT Artificial Designedpeptide based on the amino acid sequence of SEQ ID NO49 126 Phe Leu HisThr Arg Thr Pro Pro Ile 1 5 127 10 PRT Artificial Designed peptide basedon the amino acid sequence of SEQ ID NO49 127 Phe Leu His Thr Arg ThrPro Pro Ile Ile 1 5 10 128 10 PRT Artificial Designed peptide based onthe amino acid sequence of SEQ ID NO49 128 Lys Ile Gly Asp Leu Gly LeuAla Thr Leu 1 5 10 129 10 PRT Artificial Designed peptide based on theamino acid sequence of SEQ ID NO49 129 Ser Val Ile Gly Thr Pro Glu PheMet Val 1 5 10 130 9 PRT Artificial Designed peptide based on the aminoacid sequence of SEQ ID NO49 130 Val Ile Gly Thr Pro Glu Phe Met Val 1 5131 10 PRT Artificial Designed peptide based on the amino acid sequenceof SEQ ID NO49 131 Lys Val His Asp Pro Glu Ile Lys Glu Ile 1 5 10 132 10PRT Artificial Designed peptide based on the amino acid sequence of SEQID NO49 132 Arg Leu Trp Val Glu Asp Pro Lys Lys Leu 1 5 10 133 9 PRTArtificial Designed peptide based on the amino acid sequence of SEQ IDNO49 133 Gly Ala Thr Glu Phe Thr Phe Asp Leu 1 5 134 9 PRT ArtificialDesigned peptide based on the amino acid sequence of SEQ ID NO50 134 LysLeu Gln Pro Arg Lys Glu Phe Val 1 5 135 9 PRT Artificial Designedpeptide based on the amino acid sequence of SEQ ID NO50 135 Ala Leu GlyAla Leu Ala Ala Ala Leu 1 5 136 9 PRT Artificial Designed peptide basedon the amino acid sequence of SEQ ID NO50 136 Arg Leu Gly Ala Ala AlaPro Arg Val 1 5 137 9 PRT Artificial Designed peptide based on the aminoacid sequence of SEQ ID NO50 137 Phe Leu Asp Cys Phe Lys Ser Tyr Val 1 5138 9 PRT Artificial Designed peptide based on the amino acid sequenceof SEQ ID NO50 138 Ile Leu Ser Glu Met Arg Ala Ser Leu 1 5 139 9 PRTArtificial Designed peptide based on the amino acid sequence of SEQ IDNO50 139 Arg Leu Thr Phe Pro Glu Gln Ser Val 1 5 140 9 PRT ArtificialDesigned peptide based on the amino acid sequence of SEQ ID NO50 140 AlaLeu Gln Phe Arg Leu Thr Ser Val 1 5 141 9 PRT Artificial Designedpeptide based on the amino acid sequence of SEQ ID NO50 141 Val Leu GlyGln Ala Gly Ser Gly Val 1 5 142 10 PRT Artificial Designed peptide basedon the amino acid sequence of SEQ ID NO50 142 Leu Leu Val Lys His TrpTyr His Gln Val 1 5 10 143 10 PRT Artificial Designed peptide based onthe amino acid sequence of SEQ ID NO50 143 Gly Leu Trp Lys Glu Thr LeuPro Pro Val 1 5 10 144 10 PRT Artificial Designed peptide based on theamino acid sequence of SEQ ID NO50 144 Thr Leu Pro Pro Val Tyr Ala LeuGlu Leu 1 5 10 145 9 PRT Artificial Designed peptide based on the aminoacid sequence of SEQ ID NO50 145 Ser Leu Ala Glu Gly Leu Arg Thr Val 1 5146 9 PRT Artificial Designed peptide based on the amino acid sequenceof SEQ ID NO50 146 Gly Leu Ile Gln Gln His Gln His Leu 1 5 147 10 PRTArtificial Designed peptide based on the amino acid sequence of SEQ IDNO50 147 Gly Leu Pro Arg Ala Gly Cys Ser Gly Leu 1 5 10 148 10 PRTArtificial Designed peptide based on the amino acid sequence of SEQ IDNO50 148 Cys Leu His Glu Asn Cys Val His Lys Ala 1 5 10 149 9 PRTArtificial Designed peptide based on the amino acid sequence of SEQ IDNO50 149 Ile Leu Asp Glu Met Arg Ala Gln Leu 1 5 150 9 PRT ArtificialDesigned peptide based on the amino acid sequence of SEQ ID NO50 150 SerLeu Gln Phe Pro Glu Gln Asn Val 1 5 151 10 PRT Artificial Designedpeptide based on the amino acid sequence of SEQ ID NO50 151 Asn Val ProGlu Ala Leu Gln Phe Gln Leu 1 5 10 152 9 PRT Artificial Designed peptidebased on the amino acid sequence of SEQ ID NO50 152 Ser Leu Leu Pro AlaPhe Asp Ala Val 1 5 153 9 PRT Artificial Designed peptide based on theamino acid sequence of SEQ ID NO50 153 Phe Met Asn Ile Arg Pro Val LysLeu 1 5 154 10 PRT Artificial Designed peptide based on the amino acidsequence of SEQ ID NO50 154 Leu Leu Val Lys His Trp Tyr Arg Gln Val 1 510 155 10 PRT Artificial Designed peptide based on the amino acidsequence of SEQ ID NO50 155 Ser Leu Pro Pro Ala Tyr Ala Leu Glu Leu 1 510 156 9 PRT Artificial Designed peptide based on the amino acidsequence of SEQ ID NO50 156 Gly Leu Val Gln Gln His Gln Gln Leu 1 5 1579 PRT Artificial Designed peptide based on the amino acid sequence ofSEQ ID NO50 157 Leu Leu Ala Gln Glu Ala Ala Ala Leu 1 5 158 9 PRTArtificial Designed peptide based on the amino acid sequence of SEQ IDNO50 158 Ala Leu Gly Met Gln Ala Cys Phe Leu 1 5 159 9 PRT ArtificialDesigned peptide based on the amino acid sequence of SEQ ID NO50 159 PheLeu Ser Arg Asp Gly Thr Ser Val 1 5 160 9 PRT Artificial Designedpeptide based on the amino acid sequence of SEQ ID NO50 160 Phe Leu GlnPro Asn Arg Gln Phe Leu 1 5 161 9 PRT Artificial Designed peptide basedon the amino acid sequence of SEQ ID NO50 161 Phe Leu Ala Gln Val AsnLys Ala Val 1 5 162 9 PRT Artificial Designed peptide based on the aminoacid sequence of SEQ ID NO50 162 Phe Leu Ser Cys Phe Ser Gln Phe Thr 1 5163 9 PRT Artificial Designed peptide based on the amino acid sequenceof SEQ ID NO50 163 Cys Gln Gln Glu Arg Gln Phe Glu Val 1 5 164 9 PRTArtificial Designed peptide based on the amino acid sequence of SEQ IDNO50 164 Arg Gln Phe Glu Val Lys Phe Glu Val 1 5 165 10 PRT ArtificialDesigned peptide based on the amino acid sequence of SEQ ID NO50 165 MetLeu Asp Gln Ser Val Asp Phe Asp Val 1 5 10 166 10 PRT ArtificialDesigned peptide based on the amino acid sequence of SEQ ID NO50 166 SerLeu Pro Pro Gln His Gly Leu Glu Leu 1 5 10 167 10 PRT ArtificialDesigned peptide based on the amino acid sequence of SEQ ID NO50 167 AsnMet Ala Glu Gly Phe Arg Thr Val Leu 1 5 10 168 9 PRT Artificial Designedpeptide based on the amino acid sequence of SEQ ID NO50 168 Asn Met AlaGlu Gly Phe Arg Thr Val 1 5 169 9 PRT Artificial Designed peptide basedon the amino acid sequence of SEQ ID NO50 169 Arg Gln Leu Cys Ile TyrTrp Thr Ile 1 5 170 10 PRT Artificial Designed peptide based on theamino acid sequence of SEQ ID NO50 170 Asn Leu Gly His Asn Ala Arg TrpAsp Leu 1 5 10 171 9 PRT Artificial Designed peptide based on the aminoacid sequence of SEQ ID NO50 171 Gly Ile Pro Ile Gln Pro Trp Pro Val 1 5172 10 PRT Artificial Designed peptide based on the amino acid sequenceof SEQ ID NO52 172 Lys Met Asp Asp Pro Asp Tyr Trp Arg Thr 1 5 10 173 9PRT Artificial Designed peptide based on the amino acid sequence of SEQID NO52 173 Arg Leu Thr Asp Glu Gln Val Ala Leu 1 5 174 10 PRTArtificial Designed peptide based on the amino acid sequence of SEQ IDNO52 174 Arg Leu Gln Ser Gly Gln Phe Gly Asp Val 1 5 10 175 9 PRTArtificial Designed peptide based on the amino acid sequence of SEQ IDNO52 175 Val Leu Gly Arg His Lys Met His Val 1 5 176 9 PRT ArtificialDesigned peptide based on the amino acid sequence of SEQ ID NO52 176 LysMet His Val Pro Ala Pro Lys Leu 1 5 177 9 PRT Artificial Designedpeptide based on the amino acid sequence of SEQ ID NO52 177 Tyr Leu LeuSer Glu Glu Glu Arg Leu 1 5 178 9 PRT Artificial Designed peptide basedon the amino acid sequence of SEQ ID NO52 178 Phe Leu Pro Arg Lys PhePro Ser Leu 1 5 179 9 PRT Artificial Designed peptide based on the aminoacid sequence of SEQ ID NO52 179 Phe Ile Gln Glu Arg Phe Glu Arg Cys 1 5180 10 PRT Artificial Designed peptide based on the amino acid sequenceof SEQ ID NO52 180 Ala Leu Val Tyr Arg Gly His Ser Asp Leu 1 5 10 181 9PRT Artificial Designed peptide based on the amino acid sequence of SEQID NO52 181 Leu Val Tyr Arg Gly His Ser Asp Leu 1 5 182 9 PRT ArtificialDesigned peptide based on the amino acid sequence of SEQ ID NO52 182 SerVal Ser Pro Gly Gly Gln Trp Leu 1 5 183 10 PRT Artificial Designedpeptide based on the amino acid sequence of SEQ ID NO52 183 Leu Leu AsnPro Ala Leu Gly Asp Arg Leu 1 5 10 184 10 PRT Artificial Designedpeptide based on the amino acid sequence of SEQ ID NO52 184 Val Leu AlaThr Gln Gly His Thr Gln Val 1 5 10 185 10 PRT Artificial Designedpeptide based on the amino acid sequence of SEQ ID NO52 185 Phe Leu LeuVal Ala Ser Gln Arg Ser Val 1 5 10 186 9 PRT Artificial Designed peptidebased on the amino acid sequence of SEQ ID NO52 186 Leu Leu Val Ala SerGln Arg Ser Val 1 5 187 10 PRT Artificial Designed peptide based on theamino acid sequence of SEQ ID NO52 187 Arg Leu Tyr His Leu Leu Arg GlnGlu Leu 1 5 10 188 9 PRT Artificial Designed peptide based on the aminoacid sequence of SEQ ID NO52 188 Lys Leu Met Pro Asn Cys Lys Trp Val 1 5189 9 PRT Artificial Designed peptide based on the amino acid sequenceof SEQ ID NO52 189 Lys Leu Val Trp Phe Asp Leu Asp Leu 1 5 190 9 PRTArtificial Designed peptide based on the amino acid sequence of SEQ IDNO52 190 Arg Met Leu Arg His His Lys Lys Ala 1 5 191 9 PRT ArtificialDesigned peptide based on the amino acid sequence of SEQ ID NO52 191 LeuGln Asn Pro Leu Leu Val Pro Val 1 5 192 10 PRT Artificial Designedpeptide based on the amino acid sequence of SEQ ID NO52 192 Gly Val LeuAsp Val Ile Phe His Pro Thr 1 5 10 193 9 PRT Artificial Designed peptidebased on the amino acid sequence of SEQ ID NO53 193 Phe Ile Gln Gln CysThr Asp Asp Val 1 5 194 10 PRT Artificial Designed peptide based on theamino acid sequence of SEQ ID NO53 194 Ala Met Ser Lys Arg Ser Lys PheAla Leu 1 5 10 195 10 PRT Artificial Designed peptide based on the aminoacid sequence of SEQ ID NO53 195 Ala Leu Ile Thr Trp Ile Gly Glu Asn Val1 5 10 196 9 PRT Artificial Designed peptide based on the amino acidsequence of SEQ ID NO53 196 Trp Ile Gly Glu Asn Val Ser Gly Leu 1 5 1979 PRT Artificial Designed peptide based on the amino acid sequence ofSEQ ID NO53 197 Val Gln Asn Phe Ala Lys Glu Phe Val 1 5

1. An isolated peptide consisting of an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ IDNO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27,SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO:32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ IDNO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQID NO: 42, SEQ ID NO: 43 SEQ ID and SEQ ID NO: 44:
 2. An isolatedpolypeptide consisting of an amino acid sequence selected from the groupconsisting of SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO:48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52 and SEQID NO:
 53. 3. A composition comprising one or more of peptides of claim1 or polypeptides of claim 2; and a pharmaceutically acceptable carrier.4. An anti-cancer vaccine comprising one or more of peptides of claim 1or polypeptides of claim 2; and a pharmaceutically acceptable carrier.5. The anti-cancer vaccine of claim 4, wherein said cancer is pancreaticcancer, colon cancer, or stomach cancer.
 6. A method for inducingcytotoxic T lymphocytes, comprising contacting lymphocytes with one ormore of peptides of claim 1 or polypeptides of claim
 2. 7. An isolatedpolynucleotide encoding a peptide selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ IDNO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29,SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ IDNO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 SEQID and SEQ ID NO: 44 or polypeptide selected from the group consistingof SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ IDNO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO: 53,or the complementary strand thereof.
 8. An isolated polynucleotideselected from the group consisting of SEQ ID NO: 54, SEQ ID NO: 55, SEQID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60,SEQ ID NO: 61 and SEQ ID NO: 62, or the complementary strand thereof. 9.The polynucleotide of claim 8, wherein the polypeptide induces cytotoxicT lymphocytes and/or is recognized by cytotoxic T lymphocytes.
 10. Anisolated polynucleotide that hybridizes to the polynucleotide accordingto any of claims 7 to 9 under stringent conditions, or the complementarystrand thereof.
 11. A recombinant vector comprising the polynucleotideaccording to any of claims 7 to 9 or the complementary strand thereof,or a polynucleotide that hybridizes to said polynucleotide understringent conditions, or the complementary strand thereof.
 12. Arecombinant expression vector comprising the polynucleotide according toany of claims 7 to 9 or the complementary strand thereof, or apolynucleotide that hybridizes to said polynucleotide under stringentconditions, or the complementary strand thereof.
 13. A transformanttransformed with a recombinant vector of claim
 12. 14. A transformanttransformed with a recombinant expression vector of claim
 13. 15. Amethod for producing a peptide or polypeptide, comprising culturing atransformant transformed with a recombinant expression vector thatcomprises the polynucleotide according to any of claims 7 to 9 or thecomplementary strand thereof, or a polynucleotide that hybridizes tosaid polynucleotide under stringent conditions, or the complementarystrand thereof.
 16. An antibody that immunologically recognizes thepeptide according to claim 1 or the polypeptide according to claim 2.17. A method for screening for a compound that enhances at leastrecognition of the peptide according to claim 1 by HLA-A2-restrictedcytotoxic T lymphocytes, wherein said method comprises contacting saidpeptide or the polypeptide according to claim 2, with said compound; anddetermining whether said compound enhances said recognition by measuringIFN-γ production by said cytotoxic T lymphocytes.
 18. A method forscreening for a compound that enhances at least recognition of thepeptide according to claim 1 by an HLA-A2-restricted cytotoxic Tlymphocytes, wherein said method comprises contacting HLA-A2⁺ cellswhich have been pulsed with said peptide, with said cytotoxic Tlymphocytes which recognize a complex of the peptide and HLA-A2 in thepresence or absence of a test compound; and determining whether saidtest compound enhances said recognition by measuring the IFN-γproduction by said cytotoxic T lymphocytes.
 19. A method for screeningfor a compound that enhances at least recognition of the peptideaccording to claim 1 by an HLA-A2-restricted cytotoxic T lymphocytes,wherein said method comprises contacting HLA-A2⁺ cells into which thepolynucleotide according to claim 8 have been transfected, with saidcytotoxic T lymphocytes which recognize a complex of the peptide andHLA-A2 in the presence or absence of a test compound; and determiningwhether said test compound enhance said recognition by measuring theIFN-γ production from the cytotoxic T lymphocytes.
 20. A compound thatenhances recognition of at least one of the peptide according to claim 1or the polypeptide according to claim 2 by HLA-A2-restricted cytotoxic Tlymphocytes by interacting with the same.
 21. A compound that enhancesthe expression of the polynucleotide according to any of claims 7 to 9or the complementary strand thereof by interacting with the same.
 22. Apharmaceutical composition used for cancer treatment, comprising atleast one selected from a group consisting of the peptides according toclaim 1, the polypeptides according to claim 2, the polynucleotidesaccording to any of claims 7 to 9 or the complementary strand thereof,or a polynucleotide that hybridizes to said polynucleotide understringent conditions, or the complementary strand thereof, a recombinantvector or the recombinant expression vector comprising a polynucleotideencoding said peptide or polypeptide, a transformant comprising saidrecombinant vector or recombinant expression vector, and an antibodythat immunologically recognizes said peptide or said polypeptide; and apharmaceutically acceptable carrier.
 23. A reagent kit used formeasuring quantitatively or qualitatively the peptide according to claim1 or the polypeptide according to claim 2, wherein the kit comprises atleast one selected from a group consisting of said peptide, saidpolypeptide, and an antibody that immunologically recognizes saidpeptide or polypeptide.
 24. A reagent kit used for measuringquantitatively or qualitatively the polynucleotide according to any ofclaims 7 to 9, wherein the kit comprises at least one of saidpolynucleotide, or the complementary strand thereof; and a bufferedsolution to meaure said polynucleotide.
 25. (Added) An isolatedpolypeptide consisting of an amino acid sequence selected from the groupconsisting of SEQ ID NO: 45, SEQ ID N: 46, SEQ ID NO: 47, SEQ ID NO: 49,SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO:
 53. 26.(Added) An isolated polynucleotide selected from the group consisting ofSEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO:59, SEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 62, or the complementarystrand thereof.
 27. (Added) A recombinant vector comprising thepolynucleotide according to claim 7, claim 9 or claim 26, or thecomplementary strand thereof.
 28. (Added) A recombinant expressionvector comprising the polynucleotide according to claim 7, claim 9 orclaim 26, or the complementary strand thereof.
 29. (Added) Atransformant transformed with a recombinant vector or a recombinantexpression vector comprising the polynucleotide according to 7, claim 9or claim 26, or the complementary strand thereof.
 30. (Added) A methodfor producing a peptide or polypeptide, comprising culturing atransformant transformed with a recombinant expression vector thatcomprises the polynucleotide according to claim 7, claim 9, and claim26, or the complementary strand thereof.
 31. (Added) A reagent kit formeasuring quantitatively or qualitatively the peptide according to claim1 or the polypeptide according to claim 25, wherein the kit comprises atleast one selected from a group consisting of said peptide, saidpolypeptide, and an antibody that immunologically recognizes saidpeptide or polypeptide.
 32. (Added) A reagent kit for measuringquantitatively or qualitatively the polynucleotide according to claim 7,claim 9, or claim 26, wherein the kit comprises at least one of saidpolynucleotide, or the complementary strand thereof; and a bufferedsolution to meaure said polynucleotide.
 33. A method for treating cancercomprising in vivo administering the anti-cancer vaccine according toclaim 4 to a patient afflicted with cancer.
 34. A method for treatingcancer comprising in vivo administering the anti-cancer vaccineaccording to claim 4 to a patient afflicted with cancer, in an amountsufficient to induce cytotoxic T lymphocytes in said patient whichrecognize a complex of the peptide according to claim 1 and HLA-A2 andthereby lyse cancer cells in said patient.
 35. A method for treating acancer patient comprising treating lymphocytes which have been isolatedfrom said patient with the anti-cancer vaccine according to claim 4; andadministering the thus treated lymphocytes to said patient.
 36. A methodfor treating a cancer patient comprising treating lymphocytes which havebeen isolated from said patient with the anti-cancer vaccine accordingto claim 4 in an amount sufficient to induce cytotoxic T lymphocytes insaid patient which recognize the complex of the peptide according toclaim 1 and HLA-A2 and thereby lyse cancer cells in said patient; andadministering the thus treated lymphocytes to said patient.